Hexyl salicylate

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is a single generation reproductive toxicity study with a cyclohexyl form of salicylate that addresses the potential for hexyl salicylate to induce reproductive or fertility changes. The information provides some link to demonstrate the absence of reproductive or fertility effects in other salicylates that may be used to corroborate the absence of similar effects following hexyl salicylate exposure.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

cyclohexylsalicylate

IUPAC Name:

cyclohexylsalicylate

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): "SAT 940037"

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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.


IN-LIFE DATES: From: To:

Administration / exposure

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 wereevluated 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

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (vehicle control), 60, 180, or 540 mg/kg body weight
Basis:
nominal conc.

No. of animals per sex per dose:

24/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Other:

Positive control:

Not applicable

Examinations

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.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other:]

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

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

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

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):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, 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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

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:

not examined

Details on results (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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/day.