Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data are available for hexyl salicylate; however the dataset includes a number of 1 -, 2- or 3 -generation studies performed in the rat and mouse with the read-across (category) substances methyl salicylate and cyclohexyl salicylate. A weight of evidence approach is therefore taken to this endpoint.

Link to relevant study records

Referenceopen allclose all

Endpoint:
one-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
- Age at study initiation: (P) x wks; males were ~6 weeks old and weighed 172-262 g and females were ~8 weeks old and weighed 178-221 g.
- Weight at study initiation: (P) Males: 172-262 g; Females: 178-221 g
- Fasting period before study: Not applicable
- Housing: individually housed

- Diet (e.g. ad libitum): Pelleted rodent feed ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):21.5±1.5ºC
- Humidity (%):40-70%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12
Animals were individually housed except during mating and lactation in rooms with a temperature of 21.5±1.5ºC with a relative humidity of 40-70% and a light photoperiod of 12 hours dark/12 hours light.

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
In a one-generation reproduction study, groups of male and female Crl: (WI) BR Wistar rats (24/sex/group) were administered 0 (vehicle control), 60, 180, or 540 mg/kg body weight in corn oil (dose volume 5 ml/kg bw) by gavage daily throughout gametogenesis (10 weeks for males and 2 weeks for females), mating, gestation, and lactation (up to day 21 post partum).
The development and behaviour of the F1 offspring were evaluated up to weaning.
Duration of treatment / exposure:
Gavage administration daily throughout gametogenesis (10 weeks for males and 2 weeks for females), mating, gestation, and lactation (up to day 21 post partum).
Frequency of treatment:
Daily
Details on study schedule:
No details available
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
180 mg/kg bw/day (actual dose received)
Dose / conc.:
540 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24/sex/group
Control animals:
yes, concurrent vehicle
Positive control:
Not applicable to this study type
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

OTHER:
Clinical signs (1x daily), mortality (1x daily), and body weights (males: prior to treatment, at 14 weeks and prior to euthanization; females: prior to treatment, on gestation days 0, 7, 14 and 20, and post-partum days 0 or 1, 4, 7, 14, and 21) were recorded. During gestation, dams were observed for signs of abortion, premature delivery, and difficult and prolonged parturition. Moribund animals were euthanized by CO2 asphyxiation and necropsied. Surviving F0 males were euthanized by CO2 asphyxiation 14 weeks after the start of treatment and macroscopically examined (internal viscera). Organ (testes, epididymides, seminal vesicles with coagulating gland, prostate, and liver) weights were recorded and tissues showing severe changes were preserved.
Oestrous cyclicity (parental animals):
Not assessed
Sperm parameters (parental animals):
Not performed
Litter observations:
The F1 generation was examined for the total litter size, number of live vs. dead pups, sex, clinical signs (daily), weight (days 4, 7, 14 and 21 post partum), and external abnormalities. If possible, dead pups were examined for possible defects and cause of death. Live pups were also assessed for various developmental milestones, behaviour, and neuromuscular function.


Postmortem examinations (parental animals):
Surviving F0 females were euthanized by CO2 asphyxiation after weaning at about day 21 post partum (about 12 weeks after the start of treatment) and were examined macroscopically (internal and external). Implantation sites and number of sites in the uterus were recorded. Liver weights also were recorded. Ovaries, uterus, cervix, vagina, pituitary gland, liver and tissues with abnormal changes were preserved.
Postmortem examinations (offspring):
Surviving F1 pups were euthanized by CO2 asphyxiation on or shortly after post partum day 21 and underwent internal and external macroscopic examination. Brain, heart, liver, kidneys, and spleen weights were recorded for 2 pups/sex/litter and abnormal tissues were preserved. Mating index, fertility index, conception rate, abortion rate, gestation index, pre-birth loss index, pup loss (at birth) index, cumulative pup loss index, and sex ratio were determined.
Clinical signs:
effects observed, treatment-related
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
540 mg/kg
body weight changes , clinical signs , developmental effects , liver , organ weight changes
1/24 F0 males found dead during week 5 (no apparent cause). 1/24 F0 females died during delivery (day 23; delivered 2 dead pups and rest were undelivered but appeared normally developed) and 1/24 F0 females killed in extremis on gestation day 22 (pups were delivered and showed no specific findings). Clinical signs reported included salivation (F0 males: during pre-mating and mating periods; F0 females: during pre-mating period, gestation, and lactation) and rough coat in 3/24 F0 males. F0 females also showed reduced activity, rough coat, and shutting of eyes during gestation; 2/24 F0 females also showed ataxia, stretched body posture, respiratory disorder, and general poor condition during gestation (2 F0 females that died or were killed). 5/24 F0 females had increased amount of blood in bedding during delivery (authors suggest this plus clinical signs indicative of dystocia). 1/24 F0 females did not care for pups during and after delivery. F0 males showed significant decrease in body weight gain during weeks 5-10; body weight gains in F0 females similar to controls except during 3rd week of gestation (significantly decreased) and during days 7-14 of lactation (significantly increased). Feed consumption in F0 rats similar to controls except it was significantly decreased in F0 females during lactation. Feed conversion in F0 males was significantly reduced during weeks 4, 5, 7, 8, and 9 but significantly increased in week 6 and was significantly reduced in F0 females on gestation day 20. Mating performance and fertility similar to controls. Abortion rate, gestation index, gestation length, and pre-birth loss index were similar to controls. Number of implantations in the right horn was significantly reduced (no dose-response). Macroscopic examination showed no abnormal findings in F0 rats except F0 females showing increased incidence of reddening of mucous membrane of stomach associated with white coating and apparent increase in the incidence of swelling and/or a distinct lobular pattern of the liver. Liver weights in F0 males and females were significantly increased. Necropsy of reproductive organs of F0 animals showed results similar to controls. F1 animals had a slight increase in the number of litters and pups showing hematoma up to day 10 post partum. Total and live litter size at birth and live litter size on days 4, 7, 14, and 21 post partum were significantly reduced. Number of F1 males significantly reduced on day 4, but not day 21, post partum. No significant difference (from control) in the pup loss index at birth; however, slightly decreased mean value was noted. Number of litters with dead pups also was slightly increased. Cumulative pup loss indices were significantly increased; ration of litters with dead pups to litters without dead pups differed significantly from controls; numbers of litters with 4 or more pup losses from birth to day 21 post partum also increased. Treatment-related retardation in physical development noted (pinna unfolding, eruption of upper incisors, opening of eyes) and reflex development (auditory startle reflex). Behavior (open field test) of F1 females similar to controls; significantly increased total distance travelled, duration of ambulatory and stereotyped movements noted in F1 males. Significantly reduced mean group body weights in F1 animals and significantly decreased litter weights. Macroscopic exam showed slightly increased number of litters with pups showing truncated coniform heart. Relative heart weights significantly increased in F1 males, but not females. Absolute and relative liver weights significantly decreased in F1 females. Absolute kidney and spleen weights were significantly decreased in F1 males. Absolute brain weights were significantly decreased in F1 males and females.

180 mg/kg
clinical signs , no observed adverse effect level
/24 F0 males killed in extremis during week 13 (likely due to gavage error). No F0 female deaths. Clinical signs reported included rough coat in 1/24 F0 males. Body weight gain, feed conversion, and feed consumption in F0 rats similar to controls except feed conversion in F0 males was significantly reduced in week 8. Mating performance and fertility similar to controls. Abortion rate, gestation index, gestation length, ratio of litters with dead pups to litters without dead pups, cumulative pup loss index, and pre-birth loss index were similar to controls. Total number of implantations and the number in the right horn were significantly reduced (no dose-response). Macroscopic examination showed no abnormal findings in F0 rats. Necropsy of reproductive organs of F0 animals showed results similar to controls. Number of F1 males was significantly reduced on post partum day 21. Relative liver weights significantly decreased in F1 females.

60 mg/kg
No deaths, no clinical signs. Body weight gain, feed conversion, and feed consumption in F0 rats similar to controls. Mating performance and fertility similar to controls. Abortion rate, gestation index, gestation length, ratio of litters with dead pups to litters without dead pups, cumulative pup loss index, and pre-birth loss index were similar to controls. Macroscopic examination showed no abnormal findings in F0 rats. Necropsy of reproductive organs of F0 animals showed results similar to controls. Absolute and relative liver weights significantly decreased in F1 males. Relative brain weight significantly increased in F1 males.
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
other: body weight changes , clinical signs , Developmental effects , liver , organ weight changes
Sex:
male/female
Basis for effect level:
other: body weight changes , clinical signs , developmental effects , liver , organ weight changes observed at the high dose of 540 mg/kg bw/day
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 540 mg/kg bw/d: Clinical signs reported included salivation (F0 males: during pre-mating and mating periods; F0 females: during pre-mating period, gestation, and lactation) and rough coat in 3/24 F0 males. F0 females also showed reduced activity, rough coat, and shutting of eyes during gestation; 2/24 F0 females also showed ataxia, stretched body posture, respiratory disorder, and general poor condition during gestation (2 F0 females that died or were killed).

At 180 mg/kg bw/d: clinical signs reported included rough coat in 1/24 F0 males.
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
At 540 mg/kg bw/d: 1/24 F0 males found dead during week 5 (no apparent cause). 1/24 F0 females died during delivery (day 23; delivered 2 dead pups and rest were undelivered but appeared normally developed) and 1/24 F0 females killed in extremis on gestation day 22 (pups were delivered and showed no specific findings).

At 180 mg/kg bw/d: 1/24 F0 males killed in extremis during week 13 (likely due to gavage error). No F0 female deaths.

At 60 mg/kg bw/d: no deaths
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 540 mg/kg bw/d: F0 males showed significant decrease in body weight gain during weeks 5-10; body weight gains in F0 females similar to controls except during 3rd week of gestation (significantly decreased) and during days 7-14 of lactation (significantly increased).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 540 mg/kg bw/d: feed consumption in F0 rats similar to controls except it was significantly decreased in F0 females during lactation.
At 180 mg/kg bw/d: feed conversion in F0 males was significantly reduced in week 8
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 540 mg/kg bw/d: feed conversion in F0 males was significantly reduced during weeks 4, 5, 7, 8, and 9 but significantly increased in week 6 and was significantly reduced in F0 females on gestation day 20
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 540 mg/kg bw/d: liver weights in F0 males and females were significantly increased
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 540 mg/kg bw/d: macroscopic examination showed no abnormal findings in F0 rats except F0 females showing increased incidence of reddening of mucous membrane of stomach associated with white coating and apparent increase in the incidence of swelling and/or a distinct lobular pattern of the liver. Necropsy of reproductive organs of F0 animals showed results similar to controls.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
At 540 mg/kg bw/d: F1 animals had a slight increase in the number of litters and pups showing hematoma up to day 10 post partum.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
At 540 mg/kg bw/d: no significant difference (from control) in the pup loss index at birth; however, slightly decreased mean value was noted. Number of litters with dead pups also was slightly increased. Cumulative pup loss indices were significantly increased; ratio of litters with dead pups to litters without dead pups differed significantly from controls; numbers of litters with 4 or more pup losses from birth to day 21 post partum also increased. Total and live litter size at birth and live litter size on days 4, 7, 14, and 21 post partum were significantly reduced. Number of F1 males significantly reduced on day 4, but not day 21, post partum
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 540 mg/kg bw/d: significantly reduced mean group body weights in F1 animals and significantly decreased litter weights.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 540 mg/kg bw/d: relative heart weights significantly increased in F1 males, but not females. Absolute and relative liver weights significantly decreased in F1 females. Absolute kidney and spleen weights were significantly decreased in F1 males. Absolute brain weights were significantly decreased in F1 males and females.

At 180 mg/kg bw/d: relative liver weights significantly decreased in F1 females.

At 60 mg/kg bw/d: absolute and relative liver weights were significantly decreased in F1 males. Relative brain weight was significantly increased in F1 males.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 540 mg/kg bw/d: macroscopic examinations showed a slightly increased number of litters with pups showing truncated coniform heart.
Histopathological findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
At 540 mg/kg bw/d: treatment-related retardation in physical development noted (pinna unfolding, eruption of upper incisors, opening of eyes) and reflex development (auditory startle reflex). Behaviour (open field test) of F1 females similar to controls; significantly increased total distance travelled, duration of ambulatory and stereotyped movements noted in F1 males.
Developmental immunotoxicity:
not examined
540 mg/kg
Mating performance and fertility similar to controls. Abortion rate, gestation index, gestation length, and pre-birth loss index were similar to controls. Number of implantations in the right horn was significantly reduced (no dose-response).

F1 animals had a slight increase in the number of litters and pups showing hematoma up to day 10 post partum. Total and live litter size at birth and live litter size on days 4, 7, 14, and 21 post partum were significantly reduced. Number of F1 males significantly reduced on day 4, but not day 21, post partum. No significant difference (from control) in the pup loss index at birth; however, slightly decreased mean value was noted. Number of litters with dead pups also was slightly increased. Cumulative pup loss indices were significantly increased; ration of litters with dead pups to litters without dead pups differed significantly from controls; numbers of litters with 4 or more pup losses from birth to day 21 post partum also increased.

Treatment-related retardation in physical development noted (pinna unfolding, eruption of upper incisors, opening of eyes) and reflex development (auditory startle reflex). Behaviour (open field test) of F1 females similar to controls; significantly increased total distance travelled, duration of ambulatory and stereotyped movements noted in F1 males. Significantly reduced mean group body weights in F1 animals and significantly decreased litter weights.

Macroscopic exam showed slightly increased number of litters with pups showing truncated coniform heart. Relative heart weights significantly increased in F1 males, but not females. Absolute and relative liver weights significantly decreased in F1 females. Absolute kidney and spleen weights were significantly decreased in F1 males. Absolute brain weights were significantly decreased in F1 males and females.

180 mg/kg
Abortion rate, gestation index, gestation length, ratio of litters with dead pups to litters without dead pups, cumulative pup loss index, and pre-birth loss index were similar to controls.
Total number of implantations and the number in the right horn were significantly reduced (no dose-response).
Number of F1 males was significantly reduced on post partum day 21. Relative liver weights significantly decreased in F1 females

60 mg/kg
Abortion rate, gestation index, gestation length, ratio of litters with dead pups to litters without dead pups, cumulative pup loss index, and pre-birth loss index were similar to controls.
Absolute and relative liver weights significantly decreased in F1 males. Relative brain weight significantly increased in F1 males.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
developmental neurotoxicity
Key result
Critical effects observed:
no
Reproductive effects observed:
not specified

Following administration of cyclohexyl salicylate through gametogenesis to lactation of the F0 generation, concluding with weaning of the F1 offspring on day 21 post partum, the main findings were:

F0 generation

Clinical signs in the high dose group, 540 mg/kg bw/day, included rough hair coat for males and increased salivation prior to and following treatment in both sexes.

Two male mortalities occurred, one in each group dosed at 60 or 180 mg/kg w/day. No female parental deaths occurred during the treatment phase.

Weight gain among the high dose males was reduce throughout treatment, predominantly during the second half of the pre-mating phase (weeks 5 -10). Food conversion was similarly reduced for this group in the same period.

Prior to mating there was no treatment-related effect on F0 female weight gain or food conversion values. Food consumption was not significantly different for any of the treated male or female groups for the remainder of the treatment period.

No effects attributable to treatment were evident for mating performance or fertility parameters.

No effects of treatment were apparent for the males during necropsy. The high dose male liver weights were significantly increased at termination but there was no dose-related effect at lower levels.

A number of findings were noted in the high dose females, particularly on the day of delivery, at the end of gestation. Reduced activity, rough hair coat, eyes shut, respiratory disorders or presence of blood in bedding during delivery were noted at 540 mg/kg bw/day. The findings were considered indicative of dystocia.

Two deaths also occurred in the high dose group, shortly prior to or during delivery, consistent with disorders of the dams around the point of delivery.

The high dose females also showed a marked decrease in weight gain from circa day 14 to 20 of gestation, this as reflected in reduced food conversion values at the end of gestation for this group.

Throughout pregnancy none of the treated females showed signs of aborting or premature delivery and no treatment related effects on gestation index, gestation length or numbers of implantations were evident to indicate a treatment effect.

In the high dose group, there was a slight increase in the pre-birth loss index.

No F0 dams died during lactation. The high dose dams showed a significant weight gain during this period but this was not reflected in food consumpation values and was not considered toxiclogically significant.

At necropsy of the F0 dams, reddening of the gastric mucosa was noted at increased incidence and increased degree of severity in the high dose females. This finding was associated with a white coating of the mucosa in the high dose females, although both of these findings were also observed in the control group, indicating it was not an effect of treatment. Liver weights were also increased in the high dose females.

Among the males during necropsy the incidence of swelling and/or a distinct lobular pattern on the liver was increased at 540 mg/kg bw/day

F1 generation

The number of litters with pups showing a haematoma up to day 10 post partum was slightly increased forthe high dose group (540 mg/kg bw/day). This was considered to reflect the apparent signs of dystocia in the dams of the high dose group.

Litter size was significantly reduced for the high dose group for the birth to day 21 post partum period, although no effect on sex ratio was apparent. Puploss at birth was also elevated in the high dose group and the cumulative pup loss was increased significantly for the high dose group from day 4 post partum onwards. This was reflected in the increasing number of litters with 4 or more pup losses during pre-weaning development.

Examination of the physical development and reflex responses revealed retardation of pinna-unfolding, of upper incisor eruption, of eye opening and a delayed onset of the auditory startle rssponse, all showing a treatment relationship.

Mean bodyweights for male and female pups at birth were significantly reduced in the high dose group and were reduced forthe females on day 4 post partum. Litter weights were reduced forthe high dose group throughout pre-weaning, reflecting the reduced numbers of pups per litter in this group.

Behavioural tests included an inclined plane test and various open field examinations of male pups only. A slight effect on activity was indicated in the high dose group since total distance travelled, duration of ambulatory actvity and stereotypic movements were all increased.

Necropsy of the F1 pups revealed a higher number of pups with a truncated coniform heart in the high dose group. In addition, pups of the high dose group had changes in liver, heart and brain weights apparently affectd by parental treatment with 540 mg/kg bw/day cyclohexyl salicylate.

No effects considered to indicate adverse effect on reproductive function of fertility were apparent in the 180 or 60 mg/kg bw/day groups, there were no effects on the offspring at dose levels that did not cause effects in parents.

Salicylates are associated with developmental disturbances in the offspring at doses that do not cause maternal toxicity but in this study the only effects on pup development were recorded in the high dose group, 540 mg/kg bw/day, a dose level that that also induced maternal toxicity.

Conclusions:
Administration of cyclohexyl salicylate at 180 mg/kg bw/day to a single parental generation through gametogenesis, mating, gestation and through lactation to weaning at 21 days post partum, had no adverse effects on the female reproductive function or fertility response. No adverse effects were detected for reproductive toxicity in males dosed up to 540 mg/kg bw/day. With regard to development of the F1 offspring, the maternal NOAEL had no apparent effects on the F1-generation in respect of litter responses, survival, growth and behaviour.
Executive summary:

The purpose of this study was to assess the effect of cyclohexyl salicylate on male and female rats and their offspring, when administered daily by oral gavage to the parent animals throughout gametogenesis, mating, gestation, and lactation up to day 21 postpartum at dose levels of 0, 60, 180 or 540 mg/kg bw/d. Clinical signs in the high dose group (540 mg/kg bw/d) included rough hair coat for males and increased salivation prior to and following treatment in both sexes. Two male mortalities occurred, one in each group dosed at 60 or 180 mg/kg w/day. No female parental deaths occurred during the treatment phase. Weight gain among the high dose males was reduced throughout treatment, predominantly during the second half of the pre-mating phase (Weeks 5 -10). Food conversion was similarly reduced for this group in the same period. Prior to mating there was no treatment-related effect on F0 female weight gain or food conversion values. Food consumption was not significantly different for any of the treated male or female groups for the remainder of the treatment period.

No effects attributable to treatment were evident for mating performance or fertility parameters. No effects of treatment were apparent for the males during necropsy. The high dose male liver weights were significantly increased at termination but there was no dose-related effect at lower levels. A number of findings were noted in the high dose females, particularly on the day of delivery, at the end of gestation. Reduced activity, rough hair coat, eyes shut, respiratory disorders or presence of blood in bedding during delivery were noted at 540 mg/kg bw/d. The findings were considered indicative of dystocia. Two deaths also occurred in the high dose group, shortly prior to or during delivery, consistent with disorders of the dams around the point of delivery. The high dose females also showed a marked decrease in weight gain from day 14 to 20 of gestation, this as reflected in reduced food conversion values at the end of gestation for this group. Throughout pregnancy none of the treated females showed signs of aborting or premature delivery and no treatment related effects on gestation index, gestation length or numbers of implantations were evident to indicate a treatment effect. In the high dose group, there was a slight increase in the pre-birth loss index. No F0 dams died during lactation. The high dose dams showed a significant weight gain during this period but this was not reflected in food consumption values and was not considered toxicologically significant. At necropsy of the F0 dams, reddening of the gastric mucosa was noted at increased incidence and increased degree of severity in the high dose females. This finding was associated with a white coating of the mucosa in the high dose females, although both of these findings were also observed in the control group, indicating it was not an effect of treatment. Liver weights were also increased in the high dose females. Among the males during necropsy the incidence of swelling and/or a distinct lobular pattern on the liver was increased at 540 mg/kg bw/d. 

The number of litters with pups showing a haematoma up to day 10 post partum was slightly increased for the high dose group (540 mg/kg bw/day). This was considered to reflect the apparent signs of dystocia in the dams of the high dose group. Litter size was significantly reduced for the high dose group for the birth to day 21 post partum period, although no effect on sex ratio was apparent. Pup loss at birth was also elevated in the high dose group and the cumulative pup loss was increased significantly for the high dose group from Day 4 post partum onwards. This was reflected in the increasing number of litters with 4 or more pup losses during pre-weaning development.

Examination of the physical development and reflex responses revealed retardation of pinna-unfolding, of upper incisor eruption, of eye opening and a delayed onset of the auditory startle response, all showing a treatment relationship. Mean bodyweights for male and female pups at birth were significantly reduced in the high dose group and were reduced for the females on day 4 post partum. Litter weights were reduced for the high dose group throughout pre-weaning, reflecting the reduced numbers of pups per litter in this group. Behavioural tests included an inclined plane test and various open field examinations of male pups only. A slight effect on activity was indicated in the high dose group since total distance travelled, duration of ambulatory activity and stereotypic movements were all increased.

Necropsy of the F1 pups revealed a higher number of pups with a truncated coniform heart in the high dose group. In addition, pups of the high dose group had changes in liver, heart and brain weights apparently affected by parental treatment with 540 mg/kg bw/d cyclohexyl salicylate. 

No effects considered to indicate adverse effect on reproductive function of fertility were apparent in the 180 or 60 mg/kg bw/day groups, there were no effects on the offspring at dose levels that did not cause effects in parents. Salicylates are associated with developmental disturbances in the offspring at doses that do not cause maternal toxicity but in this study the only effects on pup development were recorded in the high dose group, 540 mg/kg bw/d, a dose level that that also induced maternal toxicity.

Endpoint:
one-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
From 1984/03/16 to 1982/12/23.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
according to
Guideline:
other: US NTP continuous breeding protocol
Principles of method if other than guideline:
The study design involves co-housing for a prolonged period.
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc. (Kingston, NY).
- Age at study initiation: eleven- week old.
- Weight at study initiation: 35.6 +/- 0.76 for males and 26.3 +/- 0.66 for females.
- Fasting period before study: no data
- Housing: 2 animals per cage.
- Diet: ad libitum (purina certified pelleted Rodent chow)
- Water: ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (C°): between 20-25 °C
- Humidity (%): between 20 to 70%
- Air changes (per hr): at least 10 or more air changes per hour of HEPA-filtered air.
- Photoperiod (hrs dark / hrs light): 10 hrs dark/14 hrs light.

IN-LIFE DATES: no data
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency): every two weeks
- Mixing appropriate amounts with (Type of food): dosing solutions were formulated by mixing the test article directly into different proportions of corn oil.
- Storage temperature of food: Each formulated sample will be refrigerated prior to and between dosing. An aliquot will be removed for dosing and warmed to room temperature.


VEHICLE: corn oil
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): no data
- Purity: no data
- Source: Mazola.
Details on mating procedure:
- M/F ratio per cage:
1- For Task 2: one breeding pair will be housed per cage: 40 breeding pairs in the control group, and 20 breeding pairs per group for the three groups receiving test chemical.
2- For Task 3: one male mice, previously exposed to the MTD of the test article will be matched with control female mice, similarly one female mice previously treated with the MTD of the test article will be matched with control males. The remaining 20 control males will be randomly matched with the remaining 20 control females.
- Length of cohabitation:
1- For Task 2: 100 days
2- For Task 3: 7 days

- Proof of pregnancy: vaginal plugs.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how):
- Task 2: after 14 weeks of cohabitation, each pregnant female was isolated, housed individullay for 3 weeks.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Aliquots of various dosage formulations were sent to MRI for chemical analysis. reference aliquots were within 93 to 102 percent of the indicated MeS concentrations. These limits were considered acceptable. (The detailed reports describing analysis of various reference samples are present in Appendix III of NTP report)
Duration of treatment / exposure:
7 days prior to mating then for 14 weeks of cohabitation period and 3 weeks thereafter.
Frequency of treatment:
daily
Details on study schedule:
not performed
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
- 20/sex/dose for MeS groups
- 40/sex/dose for vehicle control group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: prior to commencing the reproductive study, a preliminary range-finding study was performed. The maximum tolerated dose (MTD) in male and female CD-1 mice was determined.

Positive control:
Not required for this study type.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: yes


DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no


Oestrous cyclicity (parental animals):
not performed
Sperm parameters (parental animals):
not performed.
Litter observations:
not performed.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, after Task 3.

- Maternal animals: All surviving animals, after Task 3.


GROSS NECROPSY: no


HISTOPATHOLOGY / ORGAN WEIGHTS: not performed.
Postmortem examinations (offspring):
not performed.
Statistics:
Analyses of variance, student-test, determination of P values and non parametric test (Kruskal-Wellis).
Reproductive indices:
- Fertility Index (%): No. Fertile/No. Cohabited X 100.
- Mating Index (%): No. with Copulatory plugs/ No. Cohabited X 100.
Offspring viability indices:
not performed
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs of toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no treatment-related mortality
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
No effects of treatment were observed
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
- There was a significant decrease (p<0.05) in the mean number of litters at 500 mg/kg bw/d
- The average number of pups per litter, the proportion of pups born alive, and mean live pup weight values were also significantly reduced in this group compared to the corresponding controls.
- There was no significant effect on any of these parameters in the remaining two dose groups (only a 3% reduction in pup weight at 250 mg/kg).

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Fertility of pairs during continuous breeding

Treatment group No. Fertile/No. cohabited Fertility Index (%)
Control 38/38 100
100 mg/kg bw 17/18 94
250 mg/kg bw 18/18 100
500 mg/kg bw 16/16 100
Conclusions:
A reproductive NOAEL of 100 mg/kg bw/d is determined for this study, based on reduced litter size and pup weights at 250 and 500 mg/kg bw/d.
Executive summary:

In this NTP study, groups of CD-1 Mice were gavaged with methyl salicylate (in corn oil) at dose levels of 100, 250 or 500 mg/kg bw/d during the 7-day pre-mating period, a 14-week cohabitation period and 3 for weeks thereafter. A significant decrease in the average number of litters and a lower proportion of pups born alive were seen at 500 mg/kg bw/d. Dose-related decreases were found in average litter size and average pup weight with the decreases being statistically significant at 500 mg/kg bw/d and marginally significant at 250 mg/kg bw/d. A reproductive NOAEL of 100 mg/kg bw/d is therefore determined for this study.

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
no data are available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study is similar to OECD 416, several currently recommended observations and parameter determinations were not performed. Notwithstanding these deficiencies, the study is acceptable (reliability: 2) for reproductive risk assessment (fertility) associated with the 3-generation study (Collins and al, 1971) and the chronic study in rat conducted by Webb and Harrisson, 1963.
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
several deficiencies in relation to OECD Guideline 416 in terms of parameters studied
Principles of method if other than guideline:
Not relevant
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data
- Diet: no data
- Water: no data
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: no data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): due to the volatility of MeS only enough diet for one week was prepared at any one time.
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data


VEHICLE: none
Details on mating procedure:
no data are available.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
none
Duration of treatment / exposure:
30 days before the first mating and then throughout the experiment.
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until 4 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 4 weeks.
- Age at mating of the mated animals in the study: 12 weeks
Remarks:
Doses / Concentrations:
0.25% and 0.5% (2500 ppm and 5000 ppm, equivalent to 357 and 714 mg/kg bw MeS, or 324 and 628 mg/kg bw as SA)
Basis:
nominal in diet
No. of animals per sex per dose:
25/sex/dose (F0)
30/sex/dose (F1)
Control animals:
yes, concurrent no treatment
Details on study design:
no data are available
Positive control:
no data
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: no


DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: no

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no


Oestrous cyclicity (parental animals):
not performed
Sperm parameters (parental animals):
not performed
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter, excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities,


GROSS EXAMINATION OF DEAD PUPS: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: no data
- Maternal animals: All surviving animals, after the last litter of each generation was weaned.


GROSS NECROPSY: not perfomed


HISTOPATHOLOGY / ORGAN WEIGHTS: not performed.
Postmortem examinations (offspring):
SACRIFICE
no data

GROSS NECROPSY
- Gross necropsy consisted of external examination.

HISTOPATHOLOGY / ORGAN WEIGTHS: no data
Statistics:
Analyses of variance, student-test and determination of P values.
Reproductive indices:
- Reproduction index: no. weaned 21 days/no. liveborn x 100.
Offspring viability indices:
1- Stillborn index: no. Stillborn/total born x100
2- Viability: no. alive 5 days /no. liveborn x100
Clinical signs:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
The conception rate was higher for the two groups on MeS than for the negative control groups.
The number of unsuccessful matings for the females of the negative control group was almost double that of the MeS groups .
The Reproduction indices were comparable to or greater than those of the controls.

Key result
Dose descriptor:
NOAEL
Effect level:
714 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
- Litter size was slightly smaller in the test groups than the control.
- The neonate death rate between birth and weaning (MeS at 0.25% and 0.5%) was lower than controls.
- The negative control group experienced a larger number of stillborn than did either of the MeS.
- Viability indices of the test groups were comparable to greater than those of the controls.

GROSS PATHOLOGY (OFFSPRING)
- no abnormalities were observed in the young of the 0.25% and 0.5% MeS groups.
- All young surviving to weaning exhibited normal development in respect to body growth, appearance and behavior.

Reproductive effects observed:
not specified

table 1: mouse mating performance

 

% females (pregnancies/ matings)

 

N (females mated twice 

2/2

1/2

0/2

first generation 

control

20

85,0

5,0

10,0

0,25%

22

72,3

27,3

0

0,50%

25

72,0

28,0

0

second generation 

control

22

9,1

72,7

18,2

0,25%

18

16,7

66,7

16,6

0,50%

16

18,9

68,8

12,4

table 2: mean litter size - birth through weaning

total born

live born

not killed at birth

alive 5 days

weaned 21 days

first generation

control

12,51

12,06

12,03

11,23

9,04

0,25%

10,68

10,58

10,53

10,03

9,06

0,50%

10,56

10,3

10,19

9,42

8,96

second generation

control

9,8

9,5

9,5

8,25

6,09

0,25%

11,5

11,5

11,5

11,2

7,95

0,50%

9,82

9,82

9,71

9,71

8,89

table 3: reproduction performance indices

stillborn

viability

lactation

reproduction

first generation 

control

3,65

93,1

80,5

75

0,25%

0,99

94,8

90,3

85,6

0,50%

2,42

91,4

95,2

87

second generation 

control

3,06

86,8

73,8

64,1

0,25%

0

97,6

70,8

69,1

0,50%

0

98,8

91,6

90,5

Conclusions:
Under the test condition, no adverse reproduction effects were noted in mice at dietary levels of 357 and 714 mg/kg bw/d methyl salicylate. A NOAEL of 714 mg/kg bw/d was therefore identified for this study.
Executive summary:

A two-generation study similar to OECD guideline 416 was conducted by Abbott and Harrisson (1978), on methyl salicylate using 25 F0 mice/sex/dose and 30 F1 mice/sex/dose. The F0 mice were administered methyl salicylate in the diet at concentrations of 0.25 or 5% (2500 or 5000 ppm; equivalent to 357 or 714 mg/kg bw/d) from 30 days before the first mating and throughout the entire study period. F0 and F1 mice were each mated twice. Reproduction parameters (total born, live born, live at 5 days and at weaning) and pup abnormalities were monitored. No effects on any parameter were reported at either dose level. Therefore, the NOAEL (all endpoints) was 714 mg/kg bw/d, the highest dose tested in the study.

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
From 1982/03/17 to 1982/12/21.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this 2-generation study, Methyl salicylate was administered to CD-1 Mice by gavage according to the NTP continuous breeding protocol at dose levels of 25, 50 and 100 mg/kg bw/d (22.5, 45 and 90 mg/kg bw/d salicylic acid equivalents) for 7 days prior to mating then for a 98 day cohabitation period and 21-day segregation periods.
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc. (Kingston, NY).
- Age at study initiation: 11 weeks.
- Weight at study initiation: 35.24 +/- 0.72 g
- Fasting period before study: no data
- Housing: for the first week, animals will be housed five per cage by sex. subsequently, the females and males from the same dose group will be randomly paired and cohabited for 100 days., one breeding pair per cage (i.e., 20 breeding pairs per dose group). Thereafter each male and female will be housed individually for a period of 20 days.
- Diet: ad libitum (purina certified pelleted Rodent chow)
- Water: ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (C°): 22
- Humidity (%): no data (relative humidity will be monitored on 31-day recording hygrothermographs (Model H311, Weathermeasure Corp, Sacramento, CA located in the animal holding rooms for this study)
- Air changes (per hr): 12 to 14 times per hour.
- Photoperiod (hrs dark / hrs light): lights in each animal room were on 0700 to 2100 hours.


IN-LIFE DATES: no data
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency): due to the volatility of MeS only enough diet for one week was prepared at any one time.
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: Each formulated sample will be refrigerated prior to and between dosing. An aliquot will be removed for dosing and warmed to room temperature.


VEHICLE: corn oil
- Amount of vehicle (if gavage): 30 mg/ml
- Lot/batch no. (if required): no data
- Purity: no data
- Source: Mazola.
Details on mating procedure:
- M/F ratio per cage:
1-Task2: one breeding pair will be housed per cage: 40 breeding pairs in the control group, and 20 breeding pairs per group for the three groups receiving test chemical.
2-Task4: a male and female/cage from different litters within treatment groups
- Length of cohabitation:
1- Task2: 100 days
2-Task4: from 1 to 7 days, depending upon when a copulatory plug was detected.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how):
1- Task2: after 98 days of cohabitation, each pregnant female was isolated, housed individullay for 21 days.
2- Task4: after 1 to 7 of cohabitation, the pairs were separated and the females were allowed to deliver their litters.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The MeS concentration will be determined by the following formula:
concentration (mg/ml) = dose level (mg/kg)/dose volume (10.0 ml/kg)
Midwest Research Institute performed the analysis of the dosage formulations, all data are present in the NTP report.
Duration of treatment / exposure:
For 7 days prior to mating then for a 98 day cohabitation period and 21-day segregation periods.
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until 4 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 3 weeks.
- Age at mating of the mated animals in the study: 11 weeks
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
- 20/sex/dose for treated groups
- 40/sex/dose for vehicle control group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: prior to commencing the reproductive study, a 14 day preliminary range-finding study was performed. The maximum tolerated dose (MTD) in male and female CD-1 mice was determined.

Positive control:
no data
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: yes


DETAILED CLINICAL OBSERVATIONS: yes

BODY WEIGHT: yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no


Oestrous cyclicity (parental animals):
not performed
Sperm parameters (parental animals):
Parameters examined in F1 male parental generations:
testis weight, epididymis weight, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
-The total number of pups/litter was adjusted by analysis of covariance.


PARAMETERS EXAMINED
The following parameters were examined in F1:
number and sex of pups, live births, stillbirths, weight gain


GROSS EXAMINATION OF DEAD PUPS: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All adult males were killed by asphixiation with CO2 and discarded at the conclusion of cohabitation in Task2 after the decision to perform Task 4 was made.
- Maternal animals: low dose females were killed by asphyxiation with CO2 and discarded at the conclusion of the 98-day cohabitation period. Mid dose adult females were killed and discarded on week 2 of Task 4. The high dose weanlings would survive until the time they were to be bred.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals (mid dose: 25 and 50 mg/kg Mes) and the F2 offspring were sacrificed at 127 +/- 8 days of age.


Statistics:
The Kruskal- Wallis analyses of variance, a Chi Square test, ANOVA test and Duncan's Multiple Range Test.
Reproductive indices:
- Fertility Index (%) = No. Fertile/ No. Cohabited X 100
- Mating Index (%) = No. with Copulatory plugs/ No. Cohabited X 100
Offspring viability indices:
No data are available.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Task 2:
- one parental male and female in the control group, one parental female in the 25 mg/kg Ms day/group, and three parental in the 100 mg/kg bw/day died prior to the conclusion of the continuous breeding phase. The random distribution of deaths across treatment groups suggested that they were not treatment-related.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
- in the F1 parental mice (Task4): the body weight for males and females were unaffected by MeS treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
-In the F1 parental mice: Sperm assessment indicated no significant difference in the % motile sperm, sperm concentration or % abnormal sperm in the cauda epididymis between male mice exposed to 0 or 100 mg/kg bw/day MeS.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
-The Fertility indices were comparable to those of the controls. One control female died between 15 and 18 weeks of study and is not included in the 18 week data. (Task2)
- The percentage of pairs with the female having a copulatory plug was 76% in the high dose pairs compared to 95% in the control pairs. The difference was not statistically significant.
- In the pairs cohabited, 17 out of 19 control females were fertile compared to 11 out of 17 high dose females. This difference was marginally significant (p = 0.083)

ORGAN WEIGHTS (PARENTAL ANIMALS):
- There were no significant differences in liver and pituitary weights between the control and 100 mg/kg MeS treatment in male and female mice.
- The brain weight was increased and reproductive tract weight was decreased in female mice given 100 mg/kg bw/day MeS. The difference between the two groups appeared to be due to biological variation, it was not considered to be of toxicological significance.

GROSS PATHOLOGY and HISTOPATHOLOGY (PARENTAL ANIMALS):
- No-treatment-related gross or histopathologic lesions were noted for the pituitary, testis, epididymis, prostate or seminal vesicles in male mice, or for the pituitary, ovary, oviduct, uterus or vagina in female mice.




Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
- No significant differences existed between the groups with respect to average number of litters per breeding pair
- Litter size was comparable to that of controls
- The proportion of pups born alive per litter was not found to differ significantly among the dose groups
- No effect on number of male pups. The high dose pairs produced a smaller number of males in the pups born alive (47% vs 55%), but the difference was not statistically significant
- Adjusted Mean pup body weights:
- at 15 weeks, mean female pup weight in the high dose group was marginally increased relative to the control
- The increased female pup weight in the high dose group was not observed at 18 weeks.
- Average pup weights in the high dose group did not differ significantly from controls.
- Unadjusted Mean pup weights:
there were no significant differences in mean pup weight.
: Average pup weight in the high dose group was comparable to the controls

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Reproductive effects observed:
not specified

Table 1. Fertility of pairs during continuous Breeding (Task 2) - Premating (1 week) and cohabitation (14 weeks) periods

Treatment group (mg/kg/day) No. Fertile/No. cohabited Fertility Index (%)
0 38/39 (b) 97
25 19/19 (c) 100
50 16/16 (d) 100
100 17/17 (e) 100

b: one male and female from different pairs died prior to the conclusion of the cohabitation period during Task 2

c: one female died prior to the conclusion of the cohabitation period during Task 2

d: two females and three males died prior to the conclusion of the premating or cohabitation period during Task 2

e: Three males died prior to the conclusion of the cohabitation period during Task 2

Table 2. Mating and fertility of pairs of Task 2 offspring: effect of continuous exposure to MeS (Task 4)

Treatment group (mg/kg/day) No. with copulatory plugs/No. cohabited Mating index (%) No. Fertile/No. cohabited Fertility Index (%)
0 18/18 (c) 95 17/19 (c) 89
100 13/17 (d) 76 11/17 (d) 65

c: one female died prior to the conclusion of Task 4

d: in the 100 mg/kg/day group only 17 males were available for mating in Task 4

Table 3. Sperm analysis for Task 2 male offspring: effect of continuous exposure to MeS (Task 4)

  Treatment Group (mg MeS/kg/day)
Parameter (Mean ± S.E.) 0 100
% Motile Sperm 90.70 ± 1.90 (20) (b) 84 ± 5.54 (17)
Sperm Concentration (No. sperm x 1000/mg caudal tissue 478.34 ± 33.45 (20) 454.54 ± 41.66 (17)
% Abnormal Sperm (c) 3.55 ± 0.44 (20) 2.99 ± 0.64 (17)
% Tailless Sperm (d) 24.43 ± 1.33 (20) 25.47 ± 2.03 (17)

b: number of observations indicated in parenthesis

c: tailless sperm not included in determination of % abnormal sperm

d: tailless sperm were found to be an artefact of the method used to prepare sperm suspensions. Maceration of the cauda epididymis appeared to cause the loss of tails from many of the spermatozoa

Conclusions:
A NOAEL of 100 mg/kg bw/d was identified for this study, in the absence of any effects of treatment.
Executive summary:

A two-generation study according to the NTP continuous breeding protocol was conducted with methyl salicylate using CD-1 mice and gavage dosing. Mice were administered methyl salicylate by gavage (in corn oil) at dose levels of 25, 50 or 100 mg/kg bw/d during the 7-day pre-mating and a 98 -day cohabitation period. Treatment was not associated with any adverse effects on fertility, number of pups/litter, percentage of live pups, or on pup weight. Necropsy of the F1 animals, reared and dosed with MeS, revealed no adverse effects on terminal body and organ weights or on sperm motility, density and morphology. A NOAEL for reproductive effects of 100 mg/kg bw/d was therefore identified for this study.

.

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
no data are available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
This study is similar to OECD 416, Several currently recommended observations and parameter determinations were not performed. Notwithstanding these deficiencies, the study is acceptable (reliability: 2) for reproductive risk assessment (fertility) associated with the 3-generation study (Collins and al, 1971) and the chronic study in rat conducted by Webb and Hansen, 1963.
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
several deficiencies in relation to OECD Guideline 416 in terms of parameters studied
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Manor Farm
- Age at study initiation: for F0: 60 days old
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually
- Diet: ad libitum (Purina Diet)
- Water: ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: no data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency): due to the volatility of MeS only enough diet for one week was prepared at any one time.
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data


VEHICLE: none
Details on mating procedure:
- M/F ratio per cage: mating was accomplished by placing a male with a female of the same test group.
- Length of cohabitation: 1 week
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
none
Duration of treatment / exposure:
60 days before the first mating and then throughout the experiment
Frequency of treatment:
daily.
Details on study schedule:
- F1 parental animals not mated until 4 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 4 weeks.
- Age at mating of the mated animals in the study: 12 weeks
Dose / conc.:
0 ppm
Remarks:
Doses / Concentrations:
0.25% and 0.5% (2500 ppm and 5000 ppm equivalent to 125 and 250 mg/kg bw MeS, or 113, 225 mg/kg bw as SA).
Basis:
nominal in diet
Dose / conc.:
2 500 ppm
Remarks:
0.25%, equivalent to 125 mg/kg bw/d (113 mg/kg bw/d salicylic acid)
Dose / conc.:
5 000 ppm
Remarks:
0.5%, equivalent to 250 mg/kg bw/d (225 mg/kg bw/d salicylic acid)
No. of animals per sex per dose:
25/sex/dose (F0)
30/sex/dose (F1)
Control animals:
yes, concurrent no treatment
Details on study design:
no data are available
Positive control:
Not required for this study type
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: no


DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: no

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no


Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter, excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities,


GROSS EXAMINATION OF DEAD PUPS: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: no data
- Maternal animals: All surviving animals, after the last litter of each generation was weaned.


GROSS NECROPSY: not perfomed


HISTOPATHOLOGY / ORGAN WEIGHTS: not performed.
Postmortem examinations (offspring):
SACRIFICE
no data

GROSS NECROPSY
- Gross necropsy consisted of external examination.

HISTOPATHOLOGY / ORGAN WEIGTHS: no data
Statistics:
Analyses of variance, t-test and determination of P values.
Reproductive indices:
- Reproduction index: no. weaned 21 days/no. liveborn x 100.
Offspring viability indices:
1- Stillborn index: no. Stillborn/total born x100
2- Viability: no. alive 5 days /no. liverborn x100
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
The mating performance of the 0.25% group was comparable to that of the negative control group. That of the 0.5% group showed a higher number of unsuccessful matings; the reproduction index was decreased at 0.5% compared to the other groups, but these findings were not statistically significant.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
- Litter size was consistently decreased for the two test groups 0.25% and 0.5% compared to control groups.
- The 0.5% MeS group exhibited a higher number of deaths between birth and PND5.
- The negative control group experienced a larger number of stillborn than did either of the MeS.
- Viability indices were decreased in the 0.5% group compared to 0.25% MeS and the negative control group. But it was not statistically significant.

GROSS PATHOLOGY (OFFSPRING)
- None of the young born in the litters of the 0.25% or 0.5% MeS groups, including the F1 and F2 litters, were observed to have any gross abnormalities. The same was true for the young produced by the negative control animals.

- All young surviving to weaning appeared normal in respect to body growth, appearance and behavior.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
125 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Critical effects observed:
no
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Early post-natal death was increased at the highest dose level of 250 mg/kg bw/d.
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
125 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Mating performance

% females (pregnancies/ matings)

 

 N (females mated twice)

2/2

1/2

0/2

first generation

control

25

60,0

32,0

8,0

0,25%

24

70,8

25,0

4,2

0,50%

23

60,9

17,4

21,7

second generation

control

28

39,3

39,3

21,4

0,25%

30

33,3

36,7

30,0

0,50%

30

33,3

43,3

23,4

Mean litter size

total born

live born

not killed at birth

alive 5 days

weaned 21 days

first generation

control

11,53

11,34

11,32

11,16

9,38

0,25%

10,48

10,38

10,33

10,23

8,85

0,50%

11,03

10,63

10,53

9,38

8,09

second generation

control

9,55

8,94

8,94

8,76

8,02

0,25%

7,1

6,94

6,94

6,81

5,81

0,50%

8,49

8,33

8,33

7,58

6,41

Reproduction performance indices

 

stillborn

viability

lactation

reproduction

first generation

control

1,6

98,4

84,1

82,7

0,25%

0,95

98,6

86,8

85,5

0,50%

3,68

88,2

86,3

76,2

second generation

control

6,35

98

91,6

89,8

0,25%

2,27

98,1

85,3

83,7

0,50%

1,08

90,9

84,6

76,9

Conclusions:
Administration of methyl salicylate at dietary concentrations of 0.25% or 0.5% resulted in reduced litter size and increased neonatal mortality.
Executive summary:

In a two-generation study (comparable to OECD 416) performed using methyl salicylate, groups of 25 Wistar rats/sex were administered methyl salicylate in the diet at concentrations of 0.25 or 5% (equivalent to 125 or 250 mg/kg bw/d) for 60 days before mating and subsequently throughout mating, gestation and lactation. Both F0 and F1 rats were each mated twice. No gross abnormalities were observed in neonates of any litter and all offspring surviving to weaning were normal in respect of growth, appearance and behaviour. At the dose of 125 mg/kg bw, the only reported effect of treatment was a decrease in litter size. At 250 mg/kg bw/d, decreases in mating performance, reproductive and viability indices were noted but these findings were not statistically significant. Deaths between birth and postnatal day 5 were increased.  In this study, The NOAEL (parental) and the NOAEL (reproduction) was 250 mg/kg bw/d, but the NOAEL (development) could not be determined, given the decrease in litter size seen at 125 and 250 mg/kg bw/d.

Endpoint:
three-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
no data are available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
This study is similar to OECD guideline 416. Several currently recommended observations and parameters determinations were not performed, adult body weight and food consumption were not measured in this study, but were stated to have been unaffected at 5000 ppm in a previous study, there are no data concerning possible effects in sex organs, corpora lutea, pre-implantation or post-implantation losses for any mating. However, no abnormalities in testes/prostate or ovaries/uterus were found in a 2 years study in rats. Oestrous cycle data and sperm morphology/function data were not measured. Notwithstanding these deficiencies, the study is acceptable (reliability: 2) for reproductive risk assessment.
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
several deficiencies in relation to OECD Guideline 416 in terms of parameters studied
Principles of method if other than guideline:
Not relevant
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Osborne-Mendel
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data
- Diet: ad libitum (Purina Laboratory Chow)
- Water: ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: no data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:



DIET PREPARATION
- Rate of preparation of diet (frequency): the diet was prepared every 14 days in a manner identical to that of Webb and Hansen (1963)
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data


VEHICLE: none
Details on mating procedure:
no data are available
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
none
Duration of treatment / exposure:
100 days before the first mating and then throughout the experiment.
Frequency of treatment:
once/day
Details on study schedule:
no data are available.
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
500 ppm
Remarks:
25 mg/mg bw/d (22.5 mg/kg bw/d salicylic acid)
Dose / conc.:
1 500 ppm
Remarks:
75 mg/mg bw/d (67.5 mg/kg bw/d salicylic acid)
Dose / conc.:
3 000 ppm
Remarks:
150 mg/mg bw/d (135 mg/kg bw/d salicylic acid)
Dose / conc.:
5 000 ppm
Remarks:
250 mg/mg bw/d (225 mg/kg bw/d salicylic acid)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, plain diet
Positive control:
Not required for this study type
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: No

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter ; excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number of pups, stillbirths, live births, presence of gross anomalies,

GROSS EXAMINATION OF DEAD PUPS: no
Postmortem examinations (parental animals):
not performed
Postmortem examinations (offspring):
SACRIFICE: no data

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations for the third generation only.

HISTOPATHOLOGY :
microscopic examination of livers and kidneys was performed.
Statistics:
the Chi-2 test was used to determine significant differences between each dose and the control for each mating in each generation.
Reproductive indices:
the fertility index (number of litters cast/number of females exposed to mating).
Offspring viability indices:
the viability index (number of liveborn/total number born)
the survival index (number alive at day 4/ number born alive)
the weaning index (adjusted number of day 21 survivors/number alive at day 4)
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS:
No clinical signs of toxicity were reported.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):

- Fertility index: no significant differences for any dose/1st generation. Appreciable decreases seen in 2nd and 3rd generations/5000 ppm.
- Average litter size/female: significant decreases were seen in the second generation in the second mating at 3000 ppm and in both mating at 5000 ppm. Although decreases were seen at 1500 ppm, they were not statistically significant because of the large variation in progeny between females within a group.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no advsere toxic effects observed at highest dose level
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No adverse effects on litter size, average number of liveborn and average number of survivors observed on examination on days 4 and 21.
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No observed significant effects
Remarks on result:
other:
Key result
Critical effects observed:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant decrease was reported in fertility index at any dose, however it was considered that there were ‘appreciable’ decreases at 250 mg/kg in F2 and F3. Significant decreases were reported in average litter size, average number of live-born progeny, average numbers of survivors to PND4 and average number of weaning in the 150 and 250 mg/kg bw/day in F2.
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no adverse effects at highest dose level tested
Remarks on result:
not determinable due to absence of adverse toxic effects
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
No statistically significant decrease was reported in fertility index at any dose, however it was considered that there were ‘appreciable’ decreases at 250 mg/kg in F2 and F3. Significant decreases were reported in average litter size, average number of live-born progeny, average numbers of survivors to PND4 and average number of weaning in the 150 and 250 mg/kg bw/day in F2.
Key result
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Supplemental study: the results obtained after addition of calcium carbonate to methyl salicylate did not differ from those obtained after administration of methyl salicylate alone.

Fertility indices of rats fed methyl salicylate for 3 generations

 dietary level (ppm)                                 
     0    500     1500     3000     5000    
 generation mating   FI (a) % (b)  FI   FI  %  FI  %  FI  %
 1  20/20 100    20/20  100   20/20  100   20/20  100   20/20  100
  2  19/19 100  20/20  100  18/19  95  19/19  100  20/20  100 
 2 1  20/20 100  19/20  95  20/20  100  19/20  95  17/20  85 
  2  19/19 100  19/20  95  19/19  100  19/20  95  10/13  77 
 3 1  20/20  100 18/20  90  18/19  95  19/20  95  17/19  89 
  2  18/20  90 16/18  89  17/19  89  15/17  88  16/19  84 

(a) Fertility Index (nb of litters cast/ nb of females exposed to mating)

(b) Percent females pregnant

Average litter size of rats fed methyl salicylate for 3 generations

 dietary level (ppm)                                 
     0    500     1500     3000     5000    
 generation mating   No. (a) Av. (b)  No. Av.    No.   Av.   No.   Av.   No.   Av.
 1 208/20 10.4  211/19  11.1   207/20 10.4  235/20  11.8  188/18 10.4
  2 213/19  11.2 232/20 11.6 228/19 12.0 238/19 12.5 198/19 10.4
 2 1  216/20 10.8 205/20 10.2 206/20  10.3 169/20  8.4 124/20 6.2 (c)
  2 226/19 11.9 204/20 10.2 189/18 10.5 187/20 9.4 (d) 86/13 6.6 (c)
 3 1 192/20 9.6 188/19 9.9 172/19 9.1 170/20 8.5 179/19 9.4
  2 197/20 9.8 191/18  10.6 163/19 8.6 132/17 7.38 172/19 9.1

(a) Total number progeny/number females exposed to mating

(b) Average litter size per female exposed to mating

(c) significant at P<0.01

(d) significant at P<0.05

Viability data for rats fed methyl salicylate for 3 generations

 dietary level (ppm)                                 

 

 

 0   

500    

1500    

3000    

5000    

 gen.

mating 

 No. (a)

Av. (b) 

VI (c, d)

No.

Av. 

VI (c, d)

  No.

  Av.

VI (c, d)

  No.

  Av.

VI (c, d)

  No.

  Av.

VI (c, d)

 1

 

208/20

10.4 

1,00

211/19

 11.1

1,00

195/20

9,8

0,94

229/20

11,4

0,97

167/18

9,3

0,88

2

213/19 

11.2

1,00

231/20

11.6

1,00

226/19

11,9

0,99

237/19

12,5

1,00

189/19

9,9

0,95

 2

 

1

 215/20

10.8

1,00

203/20

10.2

0,99

203/20

10,2

0,99

164/20

8,2 (e)

0,97

106/19

5,6 (f)

0,85

2

225/19

11.8

1,00

203/20

10.2

1,00

189/18

10,5

1,00

182/20

9,1 (e)

0,97

82/13

6,3 (f)

0,95

 3

 

1

188/20

9,4

0,98

184/19

9,7

0,98

160/19

8,4

0,93

164/20

8,2

0,96

174/19

9,2

0,97

2

196/20

9.8

1,00

186/18 

10,3

0,97

155/19

8,2

0,95

118/17

6,9

0,89

166/19

8,7

0,97

(a) Total number liveborn/number females exposed to mating

(b) Average number liveborn per female exposed to mating

(c) Viability index (no. liveborn/total no. born)

(d) Not analyzed for statistical significance

(e) significant at P<0.05

(f) significant at P<0.01

Ssurvival data of rats fed methyl salicylate for 3 generations

 dietary level (ppm)                                 

 

 

 0   

500    

1500    

3000    

5000    

 gen.

mating 

 No. (a)

Av. (b) 

SI (c, d)

No.

Av. 

SI

No.

Av. 

SI

No.

Av. 

SI

No.

Av. 

SI

 1

157/17

9,2

0,90

116/14

8,3

0,82

172/19

9,1

0,96

152/15

10,1

0,92

129/15

8,6

0,94

2

202/19

10,6

0,95

196/20

9,8

0,85

205/19

10,8

0,91

218/19

11,5

0,92

168/19

8,8

0,89

 2

1

188/20

9,4

0,87

179/20

9

0,88

190/20

9,5

0,94

123/20

6,2 (e)

0,75

82/19

4,3 (f)

0,77

2

211/19

11,1

0,94

188/20

9,4

0,93

186/18

10,3

0,98

165/20

8,2 (e)

0,91

61/13

4,7 (f)

0,74

 3

1

174/20

8,7

0,93

177/19

9,3

0,96

147/19

7,7

0,92

139/20

7

0,85

147/19

7,7

0,84

2

174/20

8,7

0,89

179/18

9,9

0,96

150/19

7,9

0,97

113/17

6,6

0,96

153/19

8,1

0,92

(a) Total number day 4 survivors / no. females exposed to mating

(b) Average number day 4 survivors per female exposed to mating

(c) Survival index (no. day 4 survivors/total no. liveborn)

(d) Not analyzed for statistical significance

(e) significant at P<0.05

(f) significant at P<0.01

Weaning data of rats fed methyl salicylate for 3 generations

 dietary level (ppm)                                 

 

 

 0   

500    

1500    

3000    

5000    

 gen.

mating 

 No. (a)

Av. (b) 

WI (c, d)

No.

Av. 

WI

No.

Av. 

WI

No.

Av. 

WI

No.

Av. 

WI

 1

154/17

9,1

0,98

114/14

8,1

0,98

172/19

9,1

1,00

151/15

10,1

0,99

129/15

8,6

1,00

2

183/19

9,6

0,91

187/20

9,4

0,95

203/19

10,7

0,99

191/19

10,1

0,88

164/19

8,6

0,98

 2

1

176/20

8,8

0,94

168/20

8,4

0,94

188/20

9,4

0,99

121/20

6 (e)

0,98

74/19

3,9 (f)

0,90

2

200/19

10,5

0,95

173/20

8,6

0,92

179/18

9,9

0,96

160/20

8,0

0,97

48/13

3,7 (f)

0,79

 3

1

170/20

8,5

0,98

172/19

9,1

0,97

146/19

7,7

0,99

122/20

6,1

0,88

137/19

7,2

0,93

2

170/20

8,5

0,97

179/18

9,9

1,00

149/19

7,8

0,99

111/17

6,5

0,98

144/19

7,6

0,94

(a) Total of no. of adjusted day 21 survivors/ no. females exposed. adjusted day 21 survivors = (no. alive at day 21)/ (no. kept at day 4) x no. alive at day 4

(b) Average number adjusted day 21 survivors per female exposed to mating

(c) Weaning index (no. adjusted day 21 survivors/ total no. alive at day 4)

(d) Not analyzed for statistical significance

(e) significant at P<0.05

(f) significant at P<0.01

Conclusions:
Under the test conditions, Methyl salicylate did not significantly reduce male or female fertility. Methyl salicylate induced developmental toxicity, adverse effects on offspring viability was observed but with no evidence of increased incidence of malformations at the doses tested. The NOAELs were identified:
NOAEL (parental): 250 mg/kg bw/day
NOAEL (reproduction): 250 mg/kg bw/day
LOAEL (development): 150 mg/kg bw/day
NOAEL (development): 75 mg/kg bw/day
Executive summary:

In a 3 -generation study, rats were fed diet containing methyl salicylate at concentrations of 500, 1500, 3000 or 5000 ppm. Dose levels are equivalent to approximately 25, 75, 150 or 250 mg/kg bw/d (~22.5, 67.5, 135, 225 mg/kg bw/d salicylic acid) for 100 days before the first mating and then throughout mating, gestation and lactation of subsequent generations. No signs of toxicity were reported at any dose level. No statistically significant decrease was reported in fertility index at any dose, however it was considered that there were ‘appreciable’ decreases at 250 mg/kg in F2 and F3. Significant decreases were reported in average litter size, average number of live-born progeny, average numbers of survivors to PND4 and average number of weaning in the 150 and 250 mg/kg bw/day in F2. No external malformations were reported in pups of any litter and necropsy of the third generation weanlings showed no significant findings.

A NOAEL (parental) of 250 mg/kg bw/d is determined for this study. A NOAEL (reproduction) of 150 mg/kg bw/d is determined, based on reduced litter size at 250 mg/kg bw/d. A NOAEL (developmental) of 75 mg/kg bw/d is determined for this study based on effects at 150 and 250 mg/kg bw/d.

Endpoint:
toxicity to reproduction
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
Not stated
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: An extensive review of available data relating to salicylate reproductive toxicity was prepared based on various literature sources, laboratory data and other reviews.
Qualifier:
no guideline required
Principles of method if other than guideline:
The review addresses studies conducted to various guideline or non-guideline protocols, to investigate the overall response to salicylate exposure in respect of animal and human reproductive and developmental effects.
GLP compliance:
not specified
Limit test:
no
Sex:
not specified
Details on test animals and environmental conditions:
No details provided
Vehicle:
not specified
Remarks:
a variety of formulations have been used in the studies reviewed in the Rhodia review
Gross pathological findings:
not examined
Other effects:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
In the multi-generation reproduction studies with methyl salicylate there are indications of embryo-foetal disruption that were not considered to be attributable to any reproductive/fertility parameters. The multi-generation studies on MeS demonstrated adverse effects on offspring viability with no evidence of increased incidence of malformations at the doses tested, nor of maternal toxicity at the foetal effect level.

No developmental toxicity study, by the oral route and in accordance with test guidelines, is available for MeS. One acceptable study using dermal administration has been reported. A petroleum-based grease containing 3% MeS was applied to the backs of rats (strain not specified) at 1, 3 or 6 g/kg/day, i.e. 30, 90 and 180 mg/kg MeS (27, 81 and 162 mg/kg as SA) on GD6-15 and undiluted at 2000 mg/kg (1800 mg/kg as SA) on GD6-9. Due to 25% maternal mortality and severe dermal irritation at the high dose level, this was reduced to 1000 mg/kg (900 mg/kg as SA) during GD10-15. No effects were reported at the lower doses, but resorption was 100% at the maternally toxic high dose.
NOAEL dermal (maternal): 180 mg/kg (162 mg/kg bw/day as SA)
LOAEL dermal (development): 1000 mg/kg (900 mg/kg bw/day as SA)
NOAEL dermal (development): 180 mg/kg (162 mg/kg bw/day as SA)

SA was administered in a study similar to OECD guideline 414 by oral gavage, to pregnant Wistar rats at 75, 150 or 300 mg/kg bw/day on GD8-14. At 300 mg/kg there was an initial bodyweight loss in the dams followed by reduced bodyweight gain. 3/15 high dose dams died and there was 100% neonatal mortality. At 150 mg/kg, there were no marked effects on maternal bodyweight gain, appearance or on food or water consumption, however increased neonatal mortality and skeletal anomalies were reported.
NOAEL (maternal): 150 mg/kg
LOAEL (development): 150 mg/kg
NOAEL (development): 75 mg/kg

SA was administered in a second study (similar to OECD guideline 414), to pregnant Wistar rats at 600, 1000, 2000 or 4000 ppm salicylic acid in the diet (30, 50, 100 or 200 mg/kg bw/day) on GD8-14. A marked reduction in food consumption and bodyweight loss was reported in high dose dams only, at the beginning of administration. 15/20 dams from each group were sacrificed on GD20, with 5/20 allowed to give birth. In the high dose group, no live foetuses were obtained from 9/15 dams, with average litter size and foetal weight being reduced in the remainder. A dose related increase in skeletal anomalies was reported at 100 and 200 mg/kg bw/day. For the dams giving birth, only one high dose animal produced live young, all of whom died by PND1.
There was no significant effect to weaning in the lower dose groups.
NOAEL (maternal): 100 mg/kg
LOAEL (development): 100 mg/kg
NOAEL (development): 50 mg/kg

Several studies have reported the effects of dosing salicylates during late gestation. In a study on rats administered NaS during GD15-20 at 25, 75 or 150 mg/kg/day or during GD20-21 at 4.2, 12.5 or 25 mg/kg/day, delayed parturition was reported in 1 dam treated at 25 mg/kg (22 mg/kg as SA) and in 2 dams treated at 150 mg/kg (131 mg/kg as SA). A dose-related increase in neonatal mortality was also reported from 12.5 mg/kg/day. Studies in A/Jax mice given either NaS or ASA at a single high dose on GD17 reported high foetal mortality with increased incidence in superficial, hepatic and gastric haemorrhage in surviving foetuses.

The effects on foetal development induced by acetyl salicylic acid are detailed in the review document attached. Effects are more extensive, more defined and more fully evaluated than for non-acetylated species. Since the test substance of interest for this dossier is non-acetylated hexyl salicylate, the ASA summaries have not been reproduced here and the reduced effects for the long chain non-acetylated ester extrapolated from comparisons with MeS, which is also less foetally toxic than ASA.

The attached review document addresses a number of studies that would not normally be considered for regulatory assessment of developmental toxicity - the studies are not compliant with guideline standards - nevertheless, the results support the assertion that developmental effects at non-maternally toxic levels are primarily an effect of free salicylic acid in conjunction with placental transfer rates, mediated by the degree of acetylation. Summaries of these studies are provided in the attached document (Section 3.2.4) but have not been reproduced here.

The well-reported developmental toxicity studies comparable to current guidelines demonstrate that in rats, at doses similar to or below those causing clear maternal toxicity in terms of reduced bodyweight gain or food consumption, ASA, SA, NaS and MeS are associated with reduced or delayed development (lower foetal weight and reduced or delayed ossification) in this species. Increases in variants and malformations were reported in non-standard studies at high dose levels by subcutaneous or intraperitoneal route or at very high dose levels by oral or dermal routes. In general, the no-effect levels and dose levels required to demonstrate developmental toxicity in rats, in terms of reduced viability or increase in developmental anomalies, are consistent across the tested salicylates. There is a slight variation in the NOAELs and/or LOAELs reported for MeS (slightly higher) and NaS (slightly lower) as compared to those reported for ASA or SA both in terms of parent compound and as free salicylate for equivalent effects.

Several developmental toxicity studies including one on ASA in rats and one on ASA in rabbits, both conducted according to current guidelines for pharmaceuticals, have demonstrated that the rabbit is considerably less sensitive than the rat to the developmental toxicity of ASA. No treatment related effects were reported in rabbit foetuses at the maternally toxic dose of 250 mg/kg, a dose which in rats caused increases in early resorptions, increased post-implantation loss, increased variations and malformations. Even at the severely maternally toxic dose of 350 mg/kg, only reduced foetal weight or reduced litter size were reported, with no evidence of increased incidence of malformations.

A consistent pattern of adverse effects in rats following exposure to several salicylates during organogenesis has been described in developmental toxicity studies. Adverse effects consist of reduced offspring viability, including decreases in litter size, number of live-born progeny, number of survivors to PND4 and number of survivors to weaning at doses not causing clear maternal toxicity, with increased numbers of developmental abnormalities at higher, toxic doses.

No such effects were reported in developmental toxicity studies in rabbits, even at severely maternally toxic dose levels. Mice appear to be less sensitive than rats. Humans may be less sensitive to salicylate exposure than are rats and the effects of acetylation have to be taken into account also when extrapolating to human risk assessment.

Administration of high doses of salicylates during late gestation has shown delayed parturition and increased neonatal mortality in rats and high foetal mortality and increased incidence in haemorrhage in living foetuses in mice. Salicylates are considered therefore to potentially adversely affect embryo/foetal development in the rat.

The attached document addresses the effects of salicylate administration on human reproductive and development outcomes. Data are only available for acetylsalicylic acid. The summaries are not repoduced here but the conclusions and their impact on classification of hexyl salicylate are summarised below.

None of the salicylates ASA, SA or the salicylate esters are classified in Annex I of Directive 1999/45/EC or of Regulation 1272/2008.
ASA in its final form as a pharmaceutical is not subject to chemical classification, but full-dose ASA has been classified for many years by the US FDA in pregnancy category C for the first and second trimester of pregnancy, and as category D for the third trimester. This is a conservative approach based on adverse
pregnancy outcomes as reported in a variety of case reports and epidemiological studies. Although ASA use in pregnancy is generally not recommended, it is accepted that there are situations where the potential benefit to mother or offspring outweigh a possible risk. Low-dose ASA has not been formally allocated to a pregnancy category by FDA.

Multi-generation and fertility studies on MeS and ASA in rats and mice demonstrated no significant adverse effects on reproductive organs, sexual performance or fertility. It is therefore concluded that according to the criteria for reproductive toxicity in Directive 67/548/EEC and Regulation 1272/2008 no classification is required for any of the salicylates for effects on sexual performance or fertility. The consistent embryo-foetal toxicity in terms of reduced offspring viability demonstrated in developmental toxicity studies in rats on ASA, SA and NaS and in multi-generation studies in rats on MeS, in the absence of marked maternal toxicity, suggests that classification of these salicylates for developmental toxicity according to the criteria in Directive 67/548/EEC and Regulation 1272/2008 could be required in the absence of mitigating factors.

In order to determine whether a developmental classification for salicylates is required, it is necessary to take into account all available information, including human data on ASA as well as animal data on SA, MeS and NaS. Reduction of the level of classification, or exemption from classification, could be justified if it is considered that rat data are not relevant for human exposure. On the other hand, confirmed findings of developmental toxicity related to ASA exposure in humans could lead to a more severe classification.

Chemicals should not be classified for reproductive toxicity where reproductive effects are produced solely as a non-specific secondary consequence of other toxic effects. Salicylates have been shown to demonstrate effects consistent with inhibition of prostaglandin synthesis in mature animals and humans as well as in developing rat foetuses. As such, this mechanism does not represent intrinsic or specific developmental toxicity. The critical effects for developmental toxicity in terms of reduced viability of offspring in rats at non-maternally toxic doses could therefore be considered to be non-specific and secondary to general
salicylate toxicity. However the mechanism of inhibition of prostaglandin synthesis is a specific property of salicylates.
The adverse developmental effects of salicylates in rats are demonstrated at doses considerably below the “limit” dose of 1000 mg/kg. Reduced viability of offspring in rats has been reported in the absence of significant maternal toxicity.

Mechanistic studies comparing the pharmacological potency of ASA and SA have shown that the pharmaceutical action of ASA via COX-1 inhibition is due to the acetylated drug itself and that SA and the salicylate esters have very low potential for COX-1 inhibition. and are therefore unlikely to cause perinatal haemorrhagic effects or specific malformations such as heart or midline defects. Non-acetylated salicylates may have some potential to cause adverse effects such as reduction in offspring viability through binding of COX-2 by free salicylate. However, since the human maximum recommended therapeutic dose of ASA (4000 mg/day, equivalent to 51 mg/kg/day as SA) has been shown to be without significant hazard for pregnant women and their offspring, it can be concluded that non-acetylated salicylates do not need to be classified as hazardous for reproduction.

Reproductive toxicity is of greatest concern when effects on reproduction are induced at doses or concentrations which are not associated with significant general toxicity. Although salicylate administration is associated with foetotoxicity in rats at doses below those causing overt toxicity to the dams in terms of the
limited parameters examined in a developmental toxicity study, the NOAELs for developmental toxicity in rats are consistent with the NOEL for MeS of 50 mg/kg bw (45.4 mg/kg bw/day as SA) for general toxicity. The LOAELs for developmental toxicity are consistent with those for general toxicity.

Salicylates therefore do not exhibit developmental toxicity at doses which would otherwise be considered to be non-toxic.
Reproductive effects observed:
not specified

The attached review document does consider the toxicokinetic differences between animals and humans. a brief summaryofthe conclusions is presented below.

Studies comparing developmental effects of ASA, SA and their metabolites show consistent results indicating that it is the free salicylate which appears to be responsible for the developmental toxicity of this class of compounds in terms of embryo-foetal abnormalities, not the products of further metabolism. The initial metabolic step of hydrolysis of ASA, SA, salicylate salts and esters to free salicylate appears to be common to all species. Differences in response to salicylate therefore cannot be considered due to any qualitative differences in metabolic pathways between species. The observed species differences in sensitivity are therefore most likely to be related to differences in bioavailability of salicylate to the foetus. This could relate to differences in maternal absorption and distribution of salicylate or to differing rates of placental transfer to the embryo/foetus.

MeS is considered to be less well absorbed and hydrolysed to a lower degree than ASA in humans compared with rats.

In rats, MeS, NaS, and SA and ASA are all rapidly absorbed following oral administration even at high concentrations. Oral administration of MeS or ASA resulted in significantly lower absorption of MeS than ASA, with total plasma salicylate from MeS at 90 minutes post administration being approximately half that derived from ASA administration. Plasma analysis in rats showed rapid hydrolysis to free salicylate for all three compounds, resulting in comparable plasma concentrations of salicylate at 60 minutes post dosing.

On the other hand in humans, hydrolysis of MeS was slower and less complete, with 30% MeS unhydrolysed at 15 minutes, and 21% at 90 minutes. While this might predict a lower potential for human developmental toxicity from MeS than ASA, SA or NaS, it does not explain the lower sensitivity of humans to ASA, compared with rats.

In order to exert a toxic effect on the embryo or foetus, salicylate must be transferred through the placenta. The anatomy and physiology of the placenta varies both between species and within species at different stages of pregnancy. Different rates of transfer of salicylate from the maternal to the embryonic/foetal circulation would lead to variable peak concentrations and variable accumulation in the embryo-foetal environment. Data on maternal versus conceptal pH gradient and plasma binding suggest that rat embryos may be exposed to a higher concentration of salicylate than those of rabbits, monkeys and humans, for equivalent external doses. Although not conclusive, this provides a plausible hypothesis to explain the higher sensitivity of rats to salicylate embryofoetal toxicity.

Reproductive toxicity is of greatest concern when effects on reproduction are induced at doses or concentrations which are not associated with significant general toxicity. Although salicylate administration is associated with foetotoxicity in rats at doses below those causing overt toxicity to the dams in terms of the limited parameters examined in a developmental toxicity study, the NOAELs for developmental toxicity in rats are consistent with the NOEL for MeS of 50 mg/kg bw (45.4 mg/kg bw/day as SA) for general toxicity.

Conclusions:
This review concludes that non-acetylated salicylates do not need to be classified as hazardous for reproduction.
Executive summary:

This review focusses primarily on demonstrating the relative effects of acetylated salicylates (ASA)compared to non-acetylated forms, using methyl salicylate as the prime comparator. For the current dossier the molecule of interest is hexyl salicylate. The conclusions drawn by comparison of methyl salicylate with ASA can be extrapolated to conclude that hexyl salicylate is highly unlikely to elicit any of the potential developmental toxicity associated with the acetylated forms. 

No significant effects on fertility were reported in the multi-generation studies in rats and mice. No significant effects on the male reproductive system were reported in multi-generation studies in rats and mice or in repeat dose general toxicity studies, although no specific studies to address this parameter were available. Mechanistic studies on acetylsalicylic acid (ASA) in male rats by intramuscular and subcutaneous routes indicate some potential for adverse effects on reproductive parameters, oral studies at therapeutically more relevant doses concluded that ASA did not affect male fertility.  Deacetylation of ASA to free salicylate, occurring rapidly following oral administration, therefore appears to decrease or eliminate toxicity of ASA to male reproduction. The adverse effects reported in the multi-generation studies are related to reduced offspring viability primarily in the rat. As such, these represent developmental toxicity not reduced fertility. There is no clear evidence to consider salicylates hazardous for fertility. The overall conclusion based on various studies with acetylated and non-acetylated salicylates and with the free acid point to the conclusion that any effects on offspring viability or survival are aspects of in utero development rather than affected by reproductive parameters or reduced parental fertility. Multi-generation and fertility studies on methyl salicylate and ASA in rats and mice demonstrated no significant adverse effects on reproductive organs, sexual performance or fertility. It is therefore concluded that according to the criteria for reproductive toxicity in Directive 67/548/EEC and Regulation 1272/2008 no classification is required for any of the salicylates for effects on sexual performance or fertility. The consistent embryofoetal toxicity in terms of reduced offspring viability demonstrated in developmental toxicity studies in rats on ASA, salicylic acid and sodium salicylate, and in multi-generation studies in rats on methyl salicylate, in the absence of marked maternal toxicity, suggests that classification of these salicylates for developmental toxicity according to the criteria in Directive 67/548/EEC and Regulation 1272/2008 could be required in the absence of mitigating factors. In order to determine whether a developmental classification for salicylates is required, it is necessary to take into account all available information, including human data on ASA as well as animal data on ASA, salicylic acid and sodium salicylate. Reduction of the level of classification, or exemption from classification, could be justified if it is considered that rat data are not relevant for human exposure. On the other hand, confirmed findings of developmental toxicity related to ASA exposure in humans could lead to a more severe classification. The relevance of the various criteria for modification of classification based on animal data has been assessed in relation to ASA and other salicylates. Chemicals should not be classified for reproductive toxicity where reproductive effects are produced solely as a non-specific secondary consequence of other toxic effects. Salicylates have been shown to demonstrate effects consistent with inhibition of prostaglandin synthesis in mature animals and humans as well as in developing rat foetuses. As such, this mechanism does not represent intrinsic or specific developmental toxicity. The critical effects for developmental toxicity in terms of reduced viability of offspring in rats at non-maternally toxic doses could therefore be considered to be non-specific and secondary to general salicylate toxicity. However the mechanism of inhibition of prostaglandin synthesis is a specific property of salicylates. It would therefore not be appropriate to exclude classification on this basis.

 

The adverse developmental effects of salicylates in rats are demonstrated at doses considerably below the limit dose of 1000 mg/kg bw/d. Reduced viability of offspring in rats has been reported in the absence of significant maternal toxicity. Exclusion from classification is therefore not appropriate on this basis. Mechanistic studies comparing the pharmacological potency of ASA and SA have shown that the pharmaceutical action of ASA via COX-1 inhibition is due to the acetylated drug itself and that SA and the salicylate esters have very low potential for COX-1 inhibition. and are therefore unlikely to cause perinatal haemorrhagic effects or specific malformations such as heart or midline defects. Non-acetylated salicylates may have some potential to cause adverse effects such as reduction in offspring viability through binding of COX-2 by free salicylate. However, since the human maximum recommended therapeutic dose of ASA (4000 mg/day, equivalent to 51 mg/kg/day as SA) has been shown to be without significant hazard for pregnant women and their offspring, it can be concluded that non-acetylated salicylates do not need to be classified as hazardous for reproduction.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat
Quality of whole database:
A number of guideline-compliant or guideline-comparable studies are available for the read-across (category) substances methyl salicylate and cyclohexyl salicylate.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No data are available for hexyl salicylate; however data are available for the read-across (category) substances cyclohexyl salicylate and methyl salicylate.

In a one-generation rat study performed with cyclohexyl salicylate (Schmidt, 1995), administration of a dose level of 540 mg/kg bw/d resulted in parental toxicity. There were no effects on fertility or mating performance; however increased perinatal mortality (associated with dystocia) and increased early postnatal development were seen. No effects were observed at dose levels of 60 or 180 mg/kg bw/d.

In a three-generation rat study performed with methyl salicylate (Collins, 1971), reduced litter size and reduced early post-natal survival was seen at the highest dietary concentration of 5000 ppm (~250 mg/kg bw/d) and also at 3000 ppm ~(~150 mg/kg bw/d). A NOAEL of 1500 ppm (~75 mg/kg bw/d) is determined for this study.

In a two-generation rat study performed with methyl salicylate (Abbot, 1978), reduced litter sizes and increased neonatal mortality were seen at both dietary concentrations tested (2500 and 5000 ppm). A NOAEL could not therefore be determined for this study. A LOAEL of 2500 ppm (125 mg/kg bw/d) was determined.

In a one-generation mouse study performed using gavage administration of methyl salicylate (NTP, 1984), a NOAEL of 100 mg/kg bw/d was determined based on reduced numbers of litters and decreased offspring viability seen at 500 mg/kg bw/d and (to a lesser extent) at 250 mg/kg bw.

In a two-generation mouse study performed using methyl salicylate (Abbot, 1978), no effects were observed at the highest dietary concentration of 5000 ppm (714 mg/kg bw/d).

In an additional two-generation mouse study performed using methyl salicylate (NTP, 1984), no effects were seen at gavage dose levels of up to 100 mg/kg bw/d.

Based on all data, an overall NOAEL of 75 mg/kg bw/d is determined for the reproductive toxicity of this group of substances. 

Effects on developmental toxicity

Description of key information

No data are available for hexyl salicylate; however a guideline-compliant study is available for the read-across (category) study cyclohexyl salicylate.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
5 May 1994 - 2 February 1996.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga GmbH, D-97633 Sulzfeld.
- Age at study initiation: 8 weeks
- Weight at study initiation: mean approximately 214g
- Fasting period before study: Not documented
- Housing: Housed individually in Makrolon Type M3 cage with standard softwood bedding.
- Diet (e.g. ad libitum): Pelleted Altromin Maintenance Diet 1324 ad libitum.
- Water (e.g. ad libitum): Community tap water from Dusseldorf ad libitum.
- Acclimation period: 5 days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25°C
- Humidity (%): 44 - 75%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light / 12 hours dark at lux units of 25 - 550.

IN-LIFE DATES: From: To: Not documented
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared daily before administration. The formulation was analysed and the concentrations of the test substance in arachis oil, DAB 10, based on the results of the determination of the HPLC method were:

0.8% = 0.4g/50ml (40 mg/kg) = 0.415 g/50ml ± 0.007
2.4% = 1.2g/50ml (120 mg/kg) = 1.260 g/50ml ± 0.014
7.2% = 3.6g/50ml (360 mg/kg) = 3.720 g/50ml ± 0.014

DIET PREPARATION
- Rate of preparation of diet (frequency): Daily
- Mixing appropriate amounts with (Type of food): Not relevant as gavage method of administration used
- Storage temperature of food: Not relevant as gavage method of administration used

VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachidis oil, DAB 10.
- Concentration in vehicle: Not documented
- Amount of vehicle (if gavage): Not documented
- Lot/batch no. (if required): Not documented
- Purity: Not documented
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test concentrations were analysed using High Performance Liquid Chromatography.
Details on mating procedure:
No information on mating procedure provided as the rats obtained from the source were primiparous time-mated females.
Duration of treatment / exposure:
GD 6-15
Frequency of treatment:
Once daily
Duration of test:
20 days post coitum
No. of animals per sex per dose:
24 female rats per dose group
Group 1: 23 pregnant rats (0mg/kg)
Group 2: 23 pregnant rats (40 mg/kg)
Group 3: 23 pregnant rats (120 mg/kg)
Group 4: 22 pregnant rats (360 mg/kg)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The rationale for dose levels was based on the results of previous toxicological results.
- Rationale for animal assignment (if not random): Animals were randomly assigned.
- Other: No additional information.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily on working days

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule: Day 0, 6, 16 and 20 post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: All maternal oragans, with emphasis on the uterus, uterine contents, position of foetuses in the uterus and the number of corpura lutea

OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: No data
- Other:
Fetal examinations:
- External examinations: Yes: all live foetuses
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett-test based on pooled variance was applied for comparison between the treated and and control groups. The Steel-test was applied when the data could not be assumed to follow a normal distribution. Fisher's exact test for 2 x 2 tables was applied if the variables could be dichotomized without loss of information (Bonferroni-Holm-corrected).
Indices:
No information provided
Historical control data:
No information provided
Clinical signs:
no effects observed
Description (incidence and severity):
No compound related symptoms were observed in any treatment groups
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No deaths occurred in any of the test groups examined
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight profiles of the pregnant females were essentially similar in all groups. Mean corrected body weight gain of the treatment groups 3 and 4 were statistically different to group 1 (the vehicle control).
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic changes were noted in the dams of groups 1 - 4
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
In test groups 2 - 4, the sum of post-implantation loss was decreased by 5%, as was the sum of total embryonic deaths.The sum of embryonic deaths was also decreased by 1% in group 3. In groups 2 and 4, the sum of total foetuses was increased by 5% and in group 3 by 1%.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No deaths occurred in any of the test groups examined. No compound related symptoms were observed in any treatment groups. Body weight profiles of the pregnant females were essentially similar in all groups. Mean corrected body weight gain of the treatment groups 3 and 4 were statistically different to group 1 (the vehicle control). No compound related differences were noted between the mean reproduction data of the test groups in comparison to the control group. In test groups 2 - 4, the sum of post-implantation loss was decreased by 5%, as was the sum of total embryonic deaths.The sum of embryonic deaths was also decreased by 1% in group 3. In groups 2 and 4, the sum of total foetuses was increased by 5% and in group 3 by 1%. These findings were considered to be incidental and in the normal range. No macroscopic changes were noted in the dams of groups 1 - 4.
Key result
Dose descriptor:
NOAEL
Effect level:
360 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
None of the visceral examinations revealed any treatment related abnormalities.

Group 1 (control) : 140 foetuses examined
9 foetuses displayed hydronephrosis
12 foetuses displayed waved ureters
9 foetuses displayed ureter dilatation
1 runt
1 foetus displayed hydrocephalous internus

Group 2 (40 mg/kg bw/d) : 152 foetuses examined
21 foetuses displayed hydronephrosis
12 foetuses displayed waved ureters
9 foetuses displayed ureter dilatation

Group 3 (120 mg/kg bw/d)
152 foetuses examined
16 foetuses displayed hydronephrosis
16 foetuses displayed waved ureters
8 foetuses displayed ureter dilatation
2 foetuses displayed gut protrusion out of the abdomen (considered an artefact)

Group 4 (360 mg/kg bw/d) : 162 foetuses examined
12 foetuses displayed hydronephrosis
2 foetuses displayed waved ureters
5 foetuses displayed ureter dilatation
1 foetus displayed haematoma periorbital
1 runt

On skeletal examination, statistically significant differences were considered to be incidental. In all treatment groups, there were no relevant findings observed when retardation was examined. Any variations observed concerning ossification and the number of ribs where not considered to be statistically significant and were incidental. Skeletal malformations were observed in one case in group 1, with irregularly placed skull bones, micrognathia, cleaved vertebrae (cervical/ thoracic/ lumbar /sacral) and non-ossification of the coccygeal vertebrae. In treatment group 3, there was one case of micrognathia and one case of partial ossification of the skull bones and non-ossification of the caudal vertebral column.
Key result
Dose descriptor:
NOAEL
Effect level:
360 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
There was no evidence of developmental toxicity in this study at dose levels of up to 360 mg/kg bw/d, which were sufficient to result in mild maternal toxicity.
Executive summary:

The developmental toxicity of cyclohexyl salicylate was investigated in the rat. Groups of 23 mated female SD rats were gavaged with the test material (in arachis oil) on Days 6-15 of gestation. Clinical condition and reaction to treatment were recorded at least once daily. B ody weight was recorded on Days 0, 6, 16 and 20 of gestation.  All surviving females were sacrificed on Day 20 of gestation and the foetuses removed by caesarean section. Necropsy was performed and macroscopic examinations of the females conducted. Live foetuses were weighed, sexed and examined for visceral and skeletal abnormalities.  There were no signs of toxicity and no mortalities observed at any dose level. Maternal weight gain was not affected by any treatment and no treatment related abnormalities were noted at necropsy.  No treatment related foetal abnormalities were observed at necropsy and there were no effects on reproductive data. Maternal and developmental NOAELs of 360 mg/kg bw/d are determined for this study in the absence of any effects of toxicological significance at the highest dose level.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
360 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A modern, guideline-compliant study is available for the read-across (category) substance cyclohexyl salicylate.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The developmental toxicity of the read-across (category) substance cyclohexyl salicylate was investigated in the rat. Groups of 23 mated female SD rats were gavaged with cyclohexyl salicylate (in arachis oil) on Days 6-15 of gestation. Clinical condition and reaction to treatment were recorded at least once daily. Body weight was recorded on Days 0, 6, 16 and 20 of gestation.  All surviving females were sacrificed on Day 20 of gestation and the foetuses removed by caesarean section. Necropsy was performed and macroscopic examinations of the females conducted. Live foetuses were weighed, sexed and examined for visceral and skeletal abnormalities.  There were no signs of toxicity and no mortalities observed at any dose level. Maternal weight gain was not affected by any treatment and no treatment related abnormalities were noted at necropsy.  No treatment related foetal abnormalities were observed at necropsy and there were no effects on reproductive data. Maternal and developmental NOAELs of 360 mg/kg bw/d are determined for this study in the absence of any effects of toxicological significance at the highest dose level.

Mode of Action Analysis / Human Relevance Framework

A review by Bingham (2010) focusses primarily on demonstrating the relative effects of acetylated salicylates (ASA)compared to non-acetylated forms, using methyl salicylate as the prime comparator. For the current dossier the molecule of interest is hexyl salicylate. The conclusions drawn by comparison of methyl salicylate with ASA can be extrapolated to conclude that hexyl salicylate is highly unlikely to elicit any of the potential developmental toxicity associated with the acetylated forms. 

No significant effects on fertility were reported in the multi-generation studies in rats and mice. No significant effects on the male reproductive system were reported in multi-generation studies in rats and mice or in repeat dose general toxicity studies, although no specific studies to address this parameter were available. Mechanistic studies on acetylsalicylic acid (ASA) in male rats by intramuscular and subcutaneous routes indicate some potential for adverse effects on reproductive parameters, oral studies at therapeutically more relevant doses concluded that ASA did not affect male fertility.  Deacetylation of ASA to free salicylate, occurring rapidly following oral administration, therefore appears to decrease or eliminate toxicity of ASA to male reproduction. The adverse effects reported in the multi-generation studies are related to reduced offspring viability primarily in the rat. As such, these represent developmental toxicity not reduced fertility. There is no clear evidence to consider salicylates hazardous for fertility. The overall conclusion based on various studies with acetylated and non-acetylated salicylates and with the free acid point to the conclusion that any effects on offspring viability or survival are aspects of in utero development rather than affected by reproductive parameters or reduced parental fertility. Multi-generation and fertility studies on methyl salicylate and ASA in rats and mice demonstrated no significant adverse effects on reproductive organs, sexual performance or fertility. It is therefore concluded that according to the criteria for reproductive toxicity in Directive 67/548/EEC and Regulation 1272/2008 no classification is required for any of the salicylates for effects on sexual performance or fertility. The consistent embryofoetal toxicity in terms of reduced offspring viability demonstrated in developmental toxicity studies in rats on ASA, salicylic acid and sodium salicylate, and in multi-generation studies in rats on methyl salicylate, in the absence of marked maternal toxicity, suggests that classification of these salicylates for developmental toxicity according to the criteria in Directive 67/548/EEC and Regulation 1272/2008 could be required in the absence of mitigating factors. In order to determine whether a developmental classification for salicylates is required, it is necessary to take into account all available information, including human data on ASA as well as animal data on ASA, salicylic acid and sodium salicylate. Reduction of the level of classification, or exemption from classification, could be justified if it is considered that rat data are not relevant for human exposure. On the other hand, confirmed findings of developmental toxicity related to ASA exposure in humans could lead to a more severe classification. The relevance of the various criteria for modification of classification based on animal data has been assessed in relation to ASA and other salicylates. Chemicals should not be classified for reproductive toxicity where reproductive effects are produced solely as a non-specific secondary consequence of other toxic effects. Salicylates have been shown to demonstrate effects consistent with inhibition of prostaglandin synthesis in mature animals and humans as well as in developing rat foetuses. As such, this mechanism does not represent intrinsic or specific developmental toxicity. The critical effects for developmental toxicity in terms of reduced viability of offspring in rats at non-maternally toxic doses could therefore be considered to be non-specific and secondary to general salicylate toxicity. However the mechanism of inhibition of prostaglandin synthesis is a specific property of salicylates. It would therefore not be appropriate to exclude classification on this basis.

 

The adverse developmental effects of salicylates in rats are demonstrated at doses considerably below the limit dose of 1000 mg/kg bw/d. Reduced viability of offspring in rats has been reported in the absence of significant maternal toxicity. Exclusion from classification is therefore not appropriate on this basis. Mechanistic studies comparing the pharmacological potency of ASA and SA have shown that the pharmaceutical action of ASA via COX-1 inhibition is due to the acetylated drug itself and that SA and the salicylate esters have very low potential for COX-1 inhibition. and are therefore unlikely to cause perinatal haemorrhagic effects or specific malformations such as heart or midline defects. Non-acetylated salicylates may have some potential to cause adverse effects such as reduction in offspring viability through binding of COX-2 by free salicylate. However, since the human maximum recommended therapeutic dose of ASA (4000 mg/day, equivalent to 51 mg/kg/day as SA) has been shown to be without significant hazard for pregnant women and their offspring, it can be concluded that non-acetylated salicylates do not need to be classified as hazardous for reproduction.

Justification for classification or non-classification

Based on the results of the studies provided, the hexyl salicylate does not require classification according to Regulation EC No. 1272/2008.