Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Key study: NOAEL 180 mg/kg bw/day; rat; OECD 415; Schmidt (1995)

Link to relevant study records

Referenceopen allclose all

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 October 1992 to 2 February 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, equivalent to a valid guidelines and the study was conducted under GLP conditions. The study was performed with test material being used to support the substance on the basis of read-across.
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland GmbH, Niederlassung Sulzfeld, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: (P) males were approximately 6 wks, females were approximately 8 weeks.
- Weight at study initiation: (P) Males: 172-262 g; Females: 178-221 g
- Housing: singly in Makrolon cages
- Diet: 'Ssniff R-Z' pelleted diet ad libitum
- Water: tap water ad libitum
- Acclimation period: 13 days (males), 12 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature: 21.5 +/- 1.5°C
- Humidity: 40-70%
- Air changes: 16 per hr
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared every second day by means of a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight (for up to 21 days)
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear, referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
An analysis of the dosing solutions (identity and concentration) was performed at commencement, at week 10 and at termination. 50 mL samples per treatment group were analysed. The test material was quantified by HPLC analysis (with external calibration). The nominal concentrations were found to be in good agreement with the measured values.
Duration of treatment / exposure:
Males: ten weeks prior to mating and throughout mating up to sacrifice (i.e. 14 weeks after treatment)

Females: two weeks prior to mating and throughout mating, gestation and lactation up to weaning on, or shortly after, day 21 post partum. Ceased on the day before sacrifice.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control (Group 1)
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
Group II
Dose / conc.:
180 mg/kg bw/day (nominal)
Remarks:
Group III
Dose / conc.:
540 mg/kg bw/day (nominal)
Remarks:
Group IV
No. of animals per sex per dose:
Twenty-four
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected in agreement with study sponsor. A 90-day toxicity study and an embryotoxicity study in rats were carried out with dose levels of 40, 120 and 360 mg/kg. During the course of the former study, increased liver weights were found in the females treated with 360 mg/kg. No effects were noted in the latter study. In order to meet the recommendations of the OECD guideline the high dose should result in toxic effects. Therefore the dose level of 540 mg/kg was selected as the high dose. The other dose levels were set to 180 and 60 mg/kg in order to obtain information about possible dose-related effects an in order to establish a clear NOEL.
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (sensory and motor behaviour, coat, urine and faecal excretion, conditions of body orifices, any signs of ill health, mortality) and twice daily (viability)

BODY WEIGHT: Yes
- Time schedule for examinations: males - weekly during pre-mating treatment, after 12, 13 and 14 weeks of treatment and at terminal sacrifice; females - weekly during pre-mating treatment; days 0, 7, 14 and 20 of gestation; dams having littered on days 0 or 1 and on 4, 7, 14 and 21 post partum.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: as for body weight except during mating.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
total litter size, live births, mortality and/or abnormal young. Pups were weighed on days 4, 7, 14 and 21 post partum. All live pups were examined throughout the pre-weaning period to determine at the age at which the following developed: pinna unfolding, hair growth, eye opening, upper incisor eruption. Testis descent was observed in male pups once at weaning. All live pups were also examined for surface righting reflex (from day 1 post partum 10 100% success within litter), auditory startle reflex (from day 12 post partum 10 100% success within litter), air righting reflex and pupil reflex (once, both between days 18 and 21 post partum). Inclined plane test and open field test were performed between days 18 and 21 post partum in at lest 10 male and 10 female pups of each group.

GROSS EXAMINATION OF DEAD PUPS:
yes, where possible.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after 14 weeks of treatment.
- Maternal animals: All surviving animals or after death of the whole litter.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera with special attention given to reproductive organs (males only) and the uterus for implantation sites (females only). The weight of the liver was determined in both sexes.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations for abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
Two male and two female pups from each litter were selected to determine brain, heart, liver, kindney and spleen weights.
Statistics:
Body weight changes, food consumption, the number of implanatations and offspring were subjected to ANOVA with a subsequent multiple range test or, if indicated group mean values were compared tby the 'Krusakal-Wallis' test and the 'Mann-Whitney U-test'.

The quotient:

([weight changes / food comsumption] x 100)

was calculated for each determination phase (food conversion ratio). Litter weights were calculated from the individual pup weights. Mean body weights, food conversion, litter and pup weights were compared by the Dunnett test (two-tailed).

Organ weights were evaluated as both absolute and relative weights (% of body weight). Dose groups were compared to the control group by the Dunnett test (two-tailed) modified according to Kramer.

Sex distribution, gestation length, the number of days until successful mating, physical and reflex development were analysed by the Kruskal-Wallis test and the Mann-Whitney U-test.

The behaviour in the inclined plane and open field tests were analysed by the Kruska-Wallis test and the Wilcoxon 2-sample test (normal approximation).

Indices were compared by an appropriate statistical method, if indicated. Where appropriate, the basic unit for all parameters was the litter.
Reproductive indices:
Mating index ([No. of positive females / No. of paired females] x 100

Fertility index ([No. of pregnant females / No. of paired females] x 100

Conception rate ([No. of pregnant females / No. of positive females] x 100)

Abortion rate ([No. of dams showing abortion / No. of pregnant dams] x 100)

Gestation index ([No. of litters with live foetuses / No. of pregnant females] x 100

Pre-birth loss index ([No. of implantations - total litter size at birth / No. of implantations] x 100

Pup loss index (at birth) ([Total litter size at birth - live litter size at birth / Total litter size at birth] x 100

Cumulative pup loss index (day x) ([Total litter size at birth - live litter size on day x / Total litter size at birth] x 100

Sex ratio ([No. of live male pups per litter / No. of liver pups per litter] x 100)
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, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-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 AND MORTALITY (PARENTAL ANIMALS)
Males:
Treatment-related findings were noted in the males of group 4 during both pre-mating treatment (i.e. weeks 1-10) as well as during and after the mating period (i.e. weeks 11-14).Salivation after treatment and in single animals, even prior to treatment, was observed. Salivation is often found during toxicological studies with oral gavage of the test material and may be considered to be a local effect.

Three high dose males showed a rough coat during both phases of treatment. Since this finding was also noted in high dose females during gestation and lactation, a treatment effect cannot be excluded although only 3/24 males were affected. This finding was also observed in one male of group 3 during each of the evaluated periods (i.e. weeks 1-10 and 11-14) but a distinct effect cannot be stated because of the low numbers affected.

Three males of the control group and one in each of groups 3 and 4 showed dermatopathia on the neck but these observations were considered to be related to struggling movements during test material administration. The dermatopathia was treated with ointment during the study, the treatment of the dermatopathia was not thought to impact upon the outcome of the study.

One male of each of groups 2 and 3 died during the treatment period which was considered to be due to probable false administration and a spontaneous death, respectively.

Females:
As in the males, salivation after treatment was noted in high dose females during the two weeks of pre-mating treatment. No females died during the pre-mating treatment.

Salivation was noted in high dose females throughout gestation. In addition a number of treatment-related findings were observed in the high dose group during the third week of gestation, particularly at the day of delivery. These findings included reduced activity, rough coat and shutting of the eyes. Moreover, ataxia and a stretched body posture, respiratory disorder and a poor general condition was noted predominantly in two females one of which died during delivery and one of which was killed in extremis on day 22 of gestation.

Five high dose females revealed an increased amount of blood in the bedding during delivery. This, in conjunction with the findings detailed above which were noted at the day of or during delivery in some animals, is considered to be indicative of dystocia. One high dose female also showed no care of pups during and after birth.

In one low dose female, rough coat was also observed at the day of delivery. However, no mid dose females and only one animal of group 2 were affected. In addition, rough coat is considered to be an unspecific clinical sign which can occur sporadically in control animals. A treatment effect can therefore not be clearly stated.

Two pre-terminal deaths occurred in group 4. One animal was killed in extremis on day 22 of gestation due to a number of clinical signs; the foetuses were alive and showed no specific findings. The other animals was found dead on day 23 of gestation after giving birth to two dead pups. The other foetuses were dead but appeared 'normally' developed. The fact that both deaths occurred shortly prior to or during delivery also suggests disorders of the dams prior to or during delivery.

Salivation was noted in group 4 throughout lactation. Moreover, a rough coat was observed in a number of females in all treated groups during the first week of lactation, in some animals during day 1 and/or 2 only. In the high dose group, this sign is in correspondence with the result of clinical observations prior to delivery. In the low and mid dose groups, no dose relationship is apparent and no corroborative findings were noted during gestation with the exception of one low dose female already showing a rough coat at the day of delivery. As rough coat is considered to be an unspecific clinical sign, a treatment effect cannot be clearly stated. The other findings cannot be attributed to treatment since no dose relationship was evident. No deaths occurred during lactation.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males:
Weight gain of high dose males was significantly reduced during the second half of pre-mating (i.e. weeks 5-10). A slight, but non-significant, decrease can also be stated for the entire pre-mating treatment (i.e. weeks 0-10) and the entire treatment period (i.e. weeks 0-14) as well as during and after mating (i.e. weeks 10-14). Evaluation of mean body weights revealed the onset of decreased weight gain in high dose males during weeks 4 and 5 of treatment. This reduction in weight gain is considered to be treatment-related.

Females:
No significant difference in weight changes and mean body weights were observed in females during the pre-mating treatment. Weight gain of high dose females was significantly decreased during the third week of gestation (i.e. days 14-20). Due to this reduction, a slight but non-significant decreased weight gain can also be stated for the entire gestation period (i.e. days 0-20) which can be considered to be treatment-related.

Weight gain of group 4 females was significantly increased from days 7-14 and over the entire period of lactation (i.e. days 0/1-21). Mean body weights did not reveal and significant differences throughout lactation. The mean weights of group 4 females were slightly decreased at the start if lactation and slightly increased on day 21 compared to group 1 females. Thus, a probable compensation of decreased body weight at delivery may be considered. However, the number of pups per dam was increased in group 4 which possibly lead to a decreased milk secretion of these dams. This might also have resulted in an increased weight gain.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Evaluation of mating index, mating performance, fertility index and conception rate did not reveal any difference attributable to the treatment.

No females showed signs of abortion or premature delivery during the course of the study. Gestation indices were 100% in all groups. No significant differences in gestation length were found between groups.

The total number of implantations and the number in the right horn was significantly decreased in group 3. In addition, the number in the right horn was also significantly decreased in group 4. Since no dose relationship was evident in both the total number of implantations and in the distribution between the right and left horns, a treatment effect cannot be stated.

Pre-birth loss indices did not differ significantly between groups. However, the mean value of group 4 seemed to be increased in comparison either with that of the other groups or those found during historical studies. In addition, the number of females with four or more pre-birth losses was increased in the high dose group. Thus, a treatment effect on pre-birth loss can be considered at this dose level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males:
Relative liver weights were significantly increased in high dose males; this increase is considered to be treatment-related. With respect to male reproductive organs, no significant differences were found between groups. This result corresponds to those of mating performance and fertility as described above. Thus, there was no indication of a treatment effect on male reproductive function.

Females:
Absolute and relative liver weights were significantly increased in group 4 relative to the control group being in correspondence with the males. This finding is considered to be treatment-related.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Males:
No distinct treatment-related findings were noted in males.

Females:
During necropsy of females on, or shortly after, day 21 of lactation, reddening of the mucous membrane of the stomach was noted in a higher incidence in group 4 females in comparison with the other groups. Additionally, the degree of this finding seemed to be increased in group 4 i.e. predominantly moderate in group 4. Reddening of mucous membrane was associated with white coating in an increased number of females in group 4. These findings are considered to be a local effect of a high oral dose.

The incidence of swelling and/or a distinct lobular pattern of the liver seemed also to be increased in group 4 being in correspondence with liver weights. However, it should be noted that these findings were not observed with a higher incidence in males after 14 weeks of treatment although relative liver weights were also increased. None of the other findings were considered to be treatment-related.

OTHER FINDINGS (PARENTAL ANIMALS)
Food consumption/food conversion ratio:
No significant differences in food consumption were found in males during pre-mating treatment (i.e. weeks 1-10). In correspondence with body weight development, food conversion was significantly reduced in high dose males during weeks 4, 5, 7, 8 and 9 of treatment. Food consumption was significantly increased in these animals during week 6. During week 8, food conversion of mid dose males was also significantly reduced. Since these males showed similar, or even significantly increased values in comparison with the controls throughout pre-mating treatment, this difference in one one of ten weeks is not considered to be indicative of a treatment effect.

No significant differences were found in both parameters during pre-mating treatment of females. Food consumption did not differ significantly between groups throughout gestation. In correspondence with the weight development, food conversion was significantly decreased in high dose females on day 20 of gestation. Mean food consumption was decreased in group 4 females throughout lactation; the weight gain of these females was increased and the number of pups per dam decreased in this group possibly resulting in a decreased milk secretion. In addition, particularly during the last part of lactation, the pups also ate the provided food. Consequentially, the decreased food consumption is considered to be without toxicological importance.
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: General toxicological effects
Dose descriptor:
NOAEL
Effect level:
540 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Reproduction
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Reproduction
Clinical signs:
effects observed, treatment-related
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:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
Litter size:
The total and live litter size at birth was significantly reduced in group 4. Afterwards, the live litter size was also significantly reduced on days 4, 7, 14 and 21 post partum. Live litter sizes at birth and on day 4 were also below the historical range. This reduction of litter size is considered to be treatment-related and corresponds to the increased pup loss found in group 4.

Pup loss indices:
The pup loss index at birth did not reveal significant differences, but a slightly increase mean value was found in group 4. This value is above the maximal value of historical data. The number of litters with dead pups at birth was also slightly increased so that a treatment effect on pup loss at birth has to be considered.

Cumulative pup loss indices were significantly increased in group 4 from day 4 post partum onwards. The value of day 4 was distinctly above the historical values. The ratio of litters with dead pups to litters without dead pups up to day 21 post partum differed significantly from that of the controls and the number of litters with four or more pup losses from birth to day 21 post partum was also increased in group 4. Thus, this increase of pup loss in litters of high dose females is considered to be treatment-related. Moreover, total litter death occurred in two litters of this group whereas this event did not occur in other groups. One animal showed no care of pups during and after birth which most probably led to the death of all pups on day 1 post partum.

Cumulative pup loss indices of other groups did not differ significantly and were clearly within the range of historical data. The ratio of litters with dead pups to litters without dead pups also did not reveal significant difference in these groups. Thus, no effect on offspring viability can be determined in groups 2 and 3.

CLINICAL SIGNS (OFFSPRING)
Physical development:
Examination of physical development of live pups revealed treatment-related retardations in group 4. Pinna unfolding was significantly delayed on days 2 to 4 post partum. The cumulative frequency of pups which had obtained eruption of upper incisors was significantly reduced on days 8 to 13 post partum. The opening of the eyes revealed a significantly reduced frequency on day 14 post partum. Afterwards, a compensation is apparent in these pups, since the frequency was very similar between groups two days later i.e. day 16 post partum. This finding corresponds to the weight development of pups. The significantly decreased frequency of pups showing testis descent at weaning in group 2 is considered to be incidental since no dose relationship existed.

Reflex development:
Onset of the auditory startle reflex was significantly retarded on day 13 post partum in group 4. On day 15 post partum, the mean frequency was comparable to that of group 1. Thus, in correspondence with eye opening, this retardation was transient. Evaluation of the development of other reflexes did not reveal any treatment-related differences. But it should be noted that the cumulative frequency of pups showing the surface righting reflex was significantly increased in group 4 on day 4 post partum after having been slightly decreased on the days before.

Behavioural examinations:
No significant differences were found during the included plane test in both male and female pups. Results of the open field tests did not provide clear evidence of a treatment effect on the females since no dose related pattern is apparent. However, a significantly increased total distance travelled, duration of ambulatory and stereotyped movements were found in males of group 4. Although no distinct dose relationship is evident, an effect of treatment cannot be excluded.

BODY WEIGHT (OFFSPRING)
Mean group body weights of both make and female offspring were significantly reduced in group 4 after birth and, additionally, in the females on day 4 post partum. These decreased body weights may be associated with clinical signs which were observed in dams of this group around delivery. The fact that no significantly reduced body weights were found after day 4 post partum seems to be an indication of compensation, however, the litter size was distinctly reduced in this group. A smaller litter size usually results in an increased weight gain of pups compared with pups out of larger litters. Thus, the compensation of decreased body weights in group 4 pups may be due to decreased litter size. Litter weights were significantly decreased in group 4 from birth up to day 21 post partum. This decrease is caused by decreased pup weights but predominantly by the distinctly and significantly reduced number of pups per litter in this group.

ORGAN WEIGHTS (OFFSPRING)
Relative heart weights were significantly increased in group 4 males, whereas the weights did not differ significantly in the females. An effect of treatment of the dams on the hearts of the pups must be suspected since an increased number of hearts of a truncated coniform shape was noted in group 4.

Absolute and relative liver weights were significantly decreased in males of groups 2 and 4 and in females of group 4. Relative weights of group 3 females were also significantly reduced. No clear dose relationship is evident in the males. Thus, the difference in group 2 males cannot be attributed to treatment. Mean relative liver weight of group 3 females was only slightly, albeit significantly, reduced compared to group 1 and very similar to group 2 females. Thus, a distinct effect in group 3 cannot be stated. In group 4, a more distinct reduction was found and a slight effect may be apparent.

Correspondingly, a treatment-related, but only slight decrease in liver weight may also be stated for the males.

Absolute weights of the kidneys and spleen were significantly decreased in group 4 males. Since the terminal body weight of these animals was also significantly decreased, relative organ weights did not reveal a significant differences. Weight development of spleen and kidneys is known to correspond roughly to the weight development.

Thus, these differences in absolute weights cannot be considered to be related to the treatment of the dams.

Absolute brain weights were significantly decreased in group 4 males and females. Relative brain weights of these animals were slightly, but non-significantly increased compared to group 1. It is known that in contrast to spleen and kidneys, the brain develops primarily according to age, so the absolute weights seem to be more conclusive than the relative weights. Since both sexes were affected, a treatment relationship is considered.

The significantly increased relative brain weight of group 2 males is considered to be without biological relevance since the absolute weight was very similar to the controls.

In summary, the weights of the heart, liver and brain of group 4 pups seemed to be affected by the treatment of the parents whereas heart weights differed in the males only.

GROSS PATHOLOGY (OFFSPRING)
Necropsy of pups found dead during pre-weaning development did not reveal any specific findings which point to a manifestation of a parental treatment-effect. Macroscopic examination of pups sacrificed on, or shortly after, day 21 post partum revealed an increased number of litters with pups showing a truncated coniform heart. The number of affected pups was only slightly increased. This finding my be in correspondence with then significantly increased relative heart weights found in group 4 males. However, only two male pups but four female pups were affected in group 4 and the weights did not reveal significant differences between female groups. Even if a clear connection of the macroscopic finding with the weight was not found, an effects on the heart of the pups must be suspected at the high dose level. All other findings did not provide evidence of a treatment relationship.

OTHER FINDINGS (OFFSPRING)
Appearance and general conditions of offspring:
The number of litters and pups showing a haematoma up to day 10 post partum was slightly increased in group 4. This finding possibly corresponds to the signs of dystocia which was observed in this group. The number of pups and litters showing a hematoma up to day 10 post partum can be seen in Table 1 in 'Any other information on results incl. tables'. There was no other indication of a treatment effect on the appearance and general condition of offspring. Only single pups or certain litters of each test group showed various findings which occurred without any dose relationship.

Sex distribution/ratio:
With respect to sex distribution, the number of males was significantly reduced in group 4 on day 4 post partum. This decrease corresponds to the decreased litter size as described above. In addition, the number of males was also significantly reduced in group 3 on day 21 post partum. However, the number of makes in group 4 did not differ significantly on this day. Additionally, the sex ratio did not reveal significant differences. The percentage of males in group 1 is very high compared to historical data, therefore a treatment effect on pups of one specific sex cannot be stated.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Development e.g. litter responses, survival, growth, behaviour
Reproductive effects observed:
not specified

Table 1: Number of pups and litters showing a haematoma up to day 10 post partum

 Group  1  2  3  4
 No. of affected pups  3  1  1  8
 No. of examined litters  24  24  24  22/20
 No. of affected litters  2  1  1  5

Table 2: Summary report of effects on reproduction/development

 Group    1  2  3  4
 Pairs started  n  24  24  24  24
 Successfully mated females  n  23  23  24  24
 Successful mating days 1- 5  n  21  23  23  24
 Successful mating days 6 - 21  n  2  0  1  0
 Non-pregnant females  n  0  0  0  0
 Pregnant females, evaluated  n  24  24  24  24
 Pre-terminal deaths  n  0  0  0  2
 Abortions  n  0  0  0  0
 Gestation 21 days  n  1  4  4  0
 Gestation 22 days  n  21  19  18  20
 Gestation 23 days  n  1  0  2  2
 Dams with live young born  n  24  24  24  22
 Dams with live young at day 4  n  24  24  24  21
 Dams with live young at day 21  n  24  24  24  20
 Implantations/dam  mean  16.1  55.7  15.1*  15.7
           
 Pre-birth loss               
 (%)  mean  8.9  9.1  6.7  16.6
 Females with 0  n  6  11  14  4
 Females with 1 - 3  n  16  10  8  12
 Females with ≥4  n  2  3  2  6
           
 Pup loss               
 At birth (%)  mean  0.3  0.8  1.5  5.2
 Cumulative (day 4) (%)  mean  1.2  1.3  2.8  14.7*
 Cumulative (day 21) (%)  mean  1.9  2.2  3.6  16.5*
 From birth to day 21          
 Litters with 0  n  19  17  15  11
 Litters with 1 - 3  n  5  7  9  7
 Litters with ≥4  n  0  0  0  4
           
 Litters with pups showing a haematoma  n  2  1  1  5
           
 Live pups/litter at birth  mean  14.7  14.1  13.9  12.4*
 Live pups/litter at day 21  mean  14.5  13.9  13.5  12.1**
 Sex ratio at day 4 (% males)  mean  53.1  52.3  47.8  48.1
 Sex ratio at day 21 (% males)  mean  53.5  52.2  47.2  49.0
           
 Litter weight at birth (g)  mean  91.98  88.09  85.94  65.50***
 Litter weight at day 21 (g)  mean  578.02  544.60  544.99  448.33***
 Male pup weight at birth (g)  mean  6.48  6.45  6.41  5.66***
 Male pup weight at day 4 (g)  mean  8.96  9.00  9.13  8.52
 Male pup weight at day 21 (g)  mean  41.09  40.51  41.83  39.41
 Female pup weight at birth (g)  mean  5.99  6.03  6.01  5.15***
 Female pup weight at day 4 (g)  mean  8.59  8.58  8.75  7.70*
 Female pup weight at day 21 (g)  mean  39.36  38.83  39.99  37.62
           
 Litters with pups showing a truncated coniform heart during necropsy  n  2  1  1  6

* p < 0.05 versus group 1

** p < 0.01 versus group 1

*** p < 0.001 versus control

Table 3: Summary report of relevant findings

 Group  2  3  4
 F0 generation         
 Males         
 Salivation prior and after treatment  -  -  +
 Weight development, food conversion  -  -  ↓
 Liver weights  -  -  ↑
       
 Females         
 Salivation prior and after treatment  -  -  +
 Pre-terminal deaths at the end of gestation  -  -  ↑
 Weight development, food conversion during gestation  -  -  ↓
 Signs of dystocia  -  -  +
 Reddening of mucous membrane of the stomach with white coating  -  -  ↑
 Swelling and/or distinct locular pattern of the liver  -  -  (↑)
 Liver weights  -  -  ↑
 Pre-birth loss  -  -  ↑
       
 F1 generation         
 Litters with pups showing a haematoma up to day 10 post partum  -  -  ↑
 Litter size from birth up to day 21 post partum  -  -  ↓
 Pup loss at birth  -  -  ↑
 Cumulative pup loss up to day 21 post partum  -  -  ↑
 Litter weights from birth up to day 21 post partum  -  -  ↓
 Pup weights after birth  -  -  ↓
 Physical/reflex development  -  -  ↓
 Activity in the open field (males only)  -  -  (↑)
 Litters and pups showing a truncated coniform heart during necroscopy  -  -  (↑)
 Heart weights (males only)  -  -  ↑
 Brain and liver weights  -  -  ↓

- no relevant difference to group 1 or not observed

+ observed

↑ increased relative to group 1

↓ decreased relative to group 1

( ) suspected

Conclusions:
Under the conditions of the test, the following NOAELS were determined, 180 mg/kg for general toxicological effects on the F0 generation, 180 mg/kg for parent females and 540 mg/kg for parent males for effects on reproduction and 180 mg/kg for development of the F1 generation.
Executive summary:

The test material was administered to male and female rats and their offspring by oral gavage. The test material was administered to parent animals (F0) generation throughout gametogenesis, mating, gestation and lactation up to day 21 post partum. The development and behaviour of the offspring (F1) was evaluated up to weaning. Dose levels of 60 (group 2), 180 (group 3) and 540 (group 4) mg/kg were used. The concurrent control group (group 1) received the vehicle only.

The main findings are as follows:

F0 generation

Salivation after and in single animals, even prior to administration, was observed in males and female of the high dose group during treatment. In addition, single high dose males showed a rough coat. Two intercurrent deaths occurred in males of groups 2 and 3 which are considered to be without toxicological importance. No females died throughout pre-mating treatment.

Weight gain of male was reduced in the high dose group throughout treatment, predominantly during the second-half of the mating period, and a corresponding decrease in food conversion was found during single weeks of pre-mating treatment. Weight development and food conversion of the females were not affected during pre-mating treatment. Food consumption values did not differ significantly either between male groups during treatment or between female groups during pre-mating treatment.

Mating performance and fertility were not influenced by effects attributable to the treatment. During necropsy of the males, no treatment-related findings were observed although liver weights were significantly increased in the high dose group. A number of findings were noted in high dose females at the end of gestation, particularly at the day of delivery. These findings comprised reduced activity, rough coat, shutting of thr eyes, respiratory disorder or an increased amount of blood in the bedding during delivery. They are considered to be indicative of dystocia. Additionally, two pre-terminal deaths occurred shortly prior to or during delivery in the high dose group which also suggests disorders of the dams around delivery.

High dose females showed a significantly decreased weight gain particularly from day 14 to 20 of gestation. The food conversion ratio was significantly reduced in high dose females at the end of gestation. No females showed signs of abortion or premature delivery throughout pregnancy. Evaluation of gestation index, gestation length and number of implantations provided no evidence of treatment effect. A slight effect on pre-birth loss was found in the high dose group.

A rough coat was observed in a number of group 4 females during the first days after delivery being in correspondence with the signs observed around delivery. No females died throughout lactation. During lactation, high dose females showed a significantly increased mean weight gain. Food consumption values did not show toxicologically important differences.

Reddening of mucous membranes of the stomach was found during necropsy with an increased incidence and degree in high dose females. This finding was associated with white coating in an also increased number of high dose females. It should be noted that both findings were also observed in females of the control group. Moreover, the incidence of swelling and/or a distinct lubular pattern of the liver seemed to be increased in group 4. As in the males, liver weights were significantly increased in these females.

F1 generation

The number of litters with pups showing a hematoma up to day 10 post partum was slightly increased in group 4, most likely being in correspondence with the signs of dystocia which were observed in this group. The litter size was significantly reduced in group 4 from birth to day 21 post partum, whereby the sex ratio did not show any important difference.

Pup loss at birth was slightly increased in group 4. Cumulative pup loss was significantly increased in this group from day 4 post partum onwards, also becoming evident by an increased number of litters with four or more pup losses during pre-weaning development.

Examination of physical and reflex development revealed treatment-related retardations in pinna unfolding, upper incisor eruption, eye opening and in the onset of the auditory startle reflex. However, the delays in the last two parameters were only transient. Mean group body weights of the pups were significantly reduced in group 4 males and females at birth and in the females day 4 post partum. Litter weights were significantly decreased in the high dose group throughout pre-weaning development, predominantly caused by the distinctly reduced number of pups per litter.

Of the behavioural examinations, a slight effect on the activity of the males cannot be excluded in group 4 since the total distance travelled and the duration of the ambulatory and stereotyped movements were significantly increased.

Necropsy of the F1 pups revealed a higher number of litters with pups showing a truncated coniform heart in group 4. Additionally, the weights of the heart, liver and brain seemed to be affected in group 4 pups by the treatment of the parents, whereby differences in heart weights were found in males only.

Conclusion

The results of the study indicate the following NOAELs:

- 180 mg/kg for general toxicological effects on the F0 generation

- 180 mg/kg for parent females and 540 mg/kg for parent males for effects on reproduction

- 180 mg/kg for development of the F1 generation

Endpoint:
one-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please see 'Justification for read-across to support the REACH registration of (z)-3-hexenyl salicylate' document attached (section 13) for full details.

It is proposed to read-across to another salicylate substance in order to fulfil data requirements.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please see ''Justification for read-across to support the REACH registration of (z)-3-hexenyl salicylate' document attached for full details.

Source substance: cyclohexyl salicylate (EC 400-410-3, CAS 25485 88-5).
Target substance: (z)-3-hexenyl salicylate (EC 265-745-8, CAS 65405-77-8)

3. ANALOGUE APPROACH JUSTIFICATION
Please see 'Justification for read-across to support the REACH registration of (z)-3-hexenyl salicylate' document attached for full details.

(z)-3-hexenyl salicylate (the target substance) and the read-across substance cyclohexyl salicylate (source substance) have been characterised using the categories and databases present in the OECD QSAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. The main difference observed is structural. The target substance ((z)-3-hexenyl salicylate) contains an acyclic hexenyl chain and the source substance (cyclohexyl salicylate) contains a cyclic hexyl group.
The output from the OECD QSAR Toolbox shows that the profiles of (z)-3-hexenyl salicylate and cyclohexyl salicylate are sufficiently similar such that any available data from the source substance can be used to address the following endpoints in the REACH registration dossier for (z)-3-hexenyl salicylate:

4. DATA MATRIX
Please see 'Justification for read-across to support the REACH registration of (z)-3-hexenyl salicylate' document attached for full details.
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: General toxicological effects
Dose descriptor:
NOAEL
Effect level:
540 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Reproduction
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Reproduction
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Development e.g. litter responses, survival, growth, behaviour
Reproductive effects observed:
no
Conclusions:
Under the conditions of the test, the following NOAELS were determined, 180 mg/kg for general toxicological effects on the F0 generation, 180 mg/kg for parent females and 540 mg/kg for parent males for effects on reproduction and 180 mg/kg for development of the F1 generation.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
180 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is GLP compliant and has a Klimisch score of 2 on the basis that the test material is a read-across substance. The study is GLP compliant conducted to a standardised guideline. The study was performed on a suitable structural analogue. A supporting reproductive/developmental toxicity screen performed on the test material was also available for assessment. The overall quality of the database is therefore high.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the key study, the test material was administered to male and female rats and their offspring by oral gavage. The test material was administered to parent animals (F0) generation throughout gametogenesis, mating, gestation and lactation up to day 21 post partum. The development and behaviour of the offspring (F1) was evaluated up to weaning. Dose levels of 60 (group 2), 180 (group 3) and 540 (group 4) mg/kg were used. The concurrent control group (group 1) received the vehicle only.

The main findings are as follows:

F0generation

Salivation after and in single animals, even prior to administration, was observed in males and female of the high dose group during treatment. In addition, single high dose males showed a rough coat. Two intercurrent deaths occurred in males of groups 2 and 3 which are considered to be without toxicological importance. No females died throughout pre-mating treatment.

Weight gain of male was reduced in the high dose group throughout treatment, predominantly during the second-half of the mating period, and a corresponding decrease in food conversion was found during single weeks of pre-mating treatment. Weight development and food conversion of the females were not affected during pre-mating treatment. Food consumption values did not differ significantly either between male groups during treatment or between female groups during pre-mating treatment.

Mating performance and fertility were not influenced by effects attributable to the treatment. During necropsy of the males, no treatment-related findings were observed although liver weights were significantly increased in the high dose group. A number of findings were noted in high dose females at the end of gestation, particularly at the day of delivery. These findings comprised reduced activity, rough coat, shutting of the eyes, respiratory disorder or an increased amount of blood in the bedding during delivery. They are considered to be indicative of dystocia. Additionally, two pre-terminal deaths occurred shortly prior to or during delivery in the high dose group which also suggests disorders of the dams around delivery.

High dose females showed a significantly decreased weight gain particularly from day 14 to 20 of gestation. The food conversion ratio was significantly reduced in high dose females at the end of gestation. No females showed signs of abortion or premature delivery throughout pregnancy. Evaluation of gestation index, gestation length and number of implantations provided no evidence of treatment effect. A slight effect on pre-birth loss was found in the high dose group.

A rough coat was observed in a number of group 4 females during the first days after delivery being in correspondence with the signs observed around delivery. No females died throughout lactation. During lactation, high dose females showed a significantly increased mean weight gain. Food consumption values did not show toxicologically important differences.

Reddening of mucous membranes of the stomach was found during necropsy with an increased incidence and degree in high dose females. This finding was associated with white coating in an also increased number of high dose females. It should be noted that both findings were also observed in females of the control group. Moreover, the incidence of swelling and/or a distinct lubular pattern of the liver seemed to be increased in group 4. As in the males, liver weights were significantly increased in these females.

F1generation

The number of litters with pups showing a hematoma up to day 10 post partum was slightly increased in group 4, most likely being in correspondence with the signs of dystocia which were observed in this group. The litter size was significantly reduced in group 4 from birth to day 21 post partum, whereby the sex ratio did not show any important difference.

Pup loss at birth was slightly increased in group 4. Cumulative pup loss was significantly increased in this group from day 4 post partum onwards, also becoming evident by an increased number of litters with four or more pup losses during pre-weaning development.

Examination of physical and reflex development revealed treatment-related retardations in pinna unfolding, upper incisor eruption, eye opening and in the onset of the auditory startle reflex. However, the delays in the last two parameters were only transient. Mean group body weights of the pups were significantly reduced in group 4 males and females at birth and in the females day 4 post partum. Litter weights were significantly decreased in the high dose group throughout pre-weaning development, predominantly caused by the distinctly reduced number of pups per litter.

Of the behavioural examinations, a slight effect on the activity of the males cannot be excluded in group 4 since the total distance travelled and the duration of the ambulatory and stereotyped movements were significantly increased.

Necropsy of the F1 pups revealed a higher number of litters with pups showing a truncated coniform heart in group 4. Additionally, the weights of the heart, liver and brain seemed to be affected in group 4 pups by the treatment of the parents, whereby differences in heart weights were found in males only.

Conclusion

The results of the study indicate the following NOAELs:

- 180 mg/kg for general toxicological effects on the F0generation

- 180 mg/kg for parent females and 540 mg/kg for parent males for effects on reproduction

- 180 mg/kg for development of the F1generation

The study was performed in line with a standardised guideline and under GLP conditions and reported to a high standard. The study was performed on the read-across substance cyclohexyl salicylate, a structural analogue of the registered substance. A reliability score of 2 was therefore assigned in accordance with the principles for assessing data quality as described in Klimisch (1997).

The supporting study, dosed male and female Wistar rats with 0, 50, 200 and 700 mg/kg bw/day in a repeated dose and reproductive/developmental toxicity screen. The test material induced mortality in the high dose group and bodyweight (and bodyweight change), food consumption, haematological, clinical chemistry, microscopic and macroscopic changes in male and female rats. Specific degenerative effects were noted in the testes in the high dose group under histological examination. Effects on fertility were noticed in the females (some of which were attributed to the testicular effects). In the high dose group, none of the dams delivered any pups. Under the conditions of the test, the no-observed-adverse effect level (NOAEL) was considered to be 200 mg/kg/day for males and females for both toxicity and reproductive parameters, and the no-observed effect level (NOEL) was established as 50 mg/kg/day in both sexes. The study was performed in line with a standardised guideline and under GLP conditions and reported to a high standard. . A reliability score of 1 was therefore assigned in accordance with the principles for assessing data quality as described in Klimisch (1997).

Justification for selection of Effect on fertility via oral route:

Both available studies were performed to a high standard, however due to the limited observations performed in a screening study, the one generation reproductive toxicity study was therefore selected as the key study.

Effects on developmental toxicity

Description of key information

Key study: NOEL 360 mg/kg bw/day; rat; OECD 414, EU Method B.31; Pittermann (1996)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5th May 1994 to 31st May 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, equivalent to a valid guidelines and the study was conducted under GLP conditions. The study was performed with test material being used to support the substance on the basis of read-across.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to justification in seciton 13
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
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 Sluzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: mean approximately 214 g
- Housing: Individually in Makrolon Type M3 cage
- Diet: pelleted Altromin Maintenance Diet 1324 ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21-25 °C
- Humidity: 44-75%:
- Air changes: 10-15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
other: arachadis oil
Details on exposure:
VEHICLE
- Concentration in vehicle: 0% (group 1), 0.8% (group 2), 2.4% (group 3) and 7.2% (group 4).
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test material in the vehicle was analysed once in the study. The concentrations of the test material in the vehicle were dtermined by HPLC the concentrations found are as follows:
0.4 g/50 mL (eq. 40 mg/kg) = 0.415 g/50 mL ± 0.007
1.2 g/50 mL (eq. 120 mg/kg) = 1.260 g/50 mL ± 0.014
3.6 g/50 mL (360 mg/kg) = 3.720 g/50 mL ± 0.014
Details on mating procedure:
Mated animals were provided by the supplier.
Duration of treatment / exposure:
Days 6 to 15 of the gestation period.
Frequency of treatment:
Daily
Duration of test:
Until day 20 post coitum
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
120 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
360 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (working days)

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross macroscopic examination of all maternal organs with emphasis on the uterus, uterine contents, position of the foetuses in the uterus and the number of corpora lutea.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The following statistical methods were used:
- if the variables could be assumed to follow a normal distribution, the Dunnett-test, based on pooled variance, was applied for the comparison between the treated groups and the control group.
- the Steel-test was applied when the data could not be assumed to follow a normal distribution.
- Fisher's exact test for 2x2 tables was applied if the variables could be dichotomised without the loss of information (Bonferroni-Holm-corrected).
Indices:
Body weight gains of the maternal animals from days 0-6, 6-16, 16-20 and 6-20 post coitum were calculated.

The individual placentae and the body weight gains of the foetuses were recorded.

For the reproduction data, group mean values were calculated on a per dam basis and a percentage group basis. Only dams with live foetuses on day 20 post coitum were included in the calculations.

Mean foetal weights were calculated from the individual foetal weights on a per litter basis/group.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No deaths occurred in the controls or any of the test groups.

No treatment-related symptoms were observed in all treatment groups.

Body weights of the pregnant females were essentially similar in all groups. Mean corrected body weight gain of groups 3 and 4 were statistically different when compared to the group 1.

No treatment-related differences were noted between the mean reproduction data of the groups 2 - 4 when compared to group 1. In groups 2-4, the sum of post-implantation loss was decreased (level 5%). In groups 2 and 4, the sum of the total embryonic deaths was decreased (level 5%) and in group 3 (level 1%). In the groups 2 and 4, the sum of the total foetuses was increased (level 5%) and in the group 3 (level 1%). These findings were considered to be incidental and within the normal range.

No macroscopic changes were noted in the dams of any of the groups.
Dose descriptor:
NOEL
Effect level:
360 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Visceral malformations:
effects observed, non-treatment-related
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The weights of the live foetuses exhibited no significant differences on a litter and individual basis i.e. mean weight between the control group and the treatment groups.

The weights of the placenta showed no significant differences between the control and treatment groups. The mean value of the uteri, including content, showed an increase in groups 2 -4 (level 5%).

The sex ratio of the foetuses was not affected by treatment with the test material.

No macroscopical findings were noted at external examination of the foetuses which were considered to be an effect of the treatment with the test material. In group 1, were noted a beginning hydrops, spina bifida, exencephalia, micrognathia and one foetus with paleness. In group 3 were noted a hydrops, missing tail and one foetus with missing mandibula, no orifice and two dead foetuses. In group 4 were noted two foetuses with one common placenta.

Visceral examination:

Group 1: 140 examined foetuses
9 hydronephrosis
12 ureter waved
9 ureter dilatation
2 bronchial tree dilated
1 runt
1 hydrocephalus internus

Group 2: 152 examined foetuses
21 hydronephrosis
12 ureter waved
9 ureter dilatation

Group 3: 152 examined foetuses
16 hydronephrosis
16 ureter waved
8 ureter dilatation
2 gut protrusion our of abdomen (artefact)

Group 4: 162 foetuses examined
12 hydronephrosis
2 ureter waved
5 ureter dilatation
1 hematoma periorbital
1 runt

The visceral examination of the preserved foetuses did not reveal and treatment-related abnormalities.

Skeletal examination of foetuses:
General observations:
The statistically significant differences were considered to be incidental. The findings concerning the ossification of the foetal skeleton showed no dose-relationship i.e. there was no indication for an abnormal ossification of the foetal skeleton, skull bones, sternebrae and coccygeal vertebrae in the treated groups compared to the controls.

Retardations:
No relevant findings in any of the groups.

Variations:
The statistical analysis of the findings sternebrae, incompletely ossified, ossified or abnormally ossified, does not indicate a dose-relationship i.e. no disturbance of the ossification of the sternebrae could be observed in any of the treated groups when compared to the controls. Concerning the number of ribs, the statistically significant difference was considered to be incidental.

Malformations:
Group 1 - one case (skull bones irregular placed, micrognathia, vertebrae (cervical/thoracal/lumbal/sacral) cleaved), coccygeal vertebrae non ossified.
Group 2 - no variations
Group 3 - two cases (micrognathia; skull bones partially ossified, vertebral column caudal non ossified)
Group 4 - no variations
Dose descriptor:
NOEL
Effect level:
360 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOEL
Effect level:
360 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1: Summary of performance of mated females

 Group

 1

 2

 3

 4

 Dose (mg/kg)

 0

 40

 120

 360

 No. of mated females

24

 24

 24

 24

 No. of pregnant females

 23

 23

 23

 22

 No. of mortalities

 0

 0

 0

 0

 No. of females included in statistical analysis (females with live foetuses at termination)

 23

 23

 23

 22

 

Table 2: Mean differences in dam body weights

 Days post coitum

 

 Group (mg/kg)

 0 - 6 (% gain)

 6 - 16 (% gain)

 16 - 20 (% gain)

 6 - 20 (% gain)

 Corrected body weight gain (%)

 1 (0)

 49.0 (22.6)

 83.3 (31.4)

 56.1 916.1)

 139.3 (52.5)

 22.9

 2 (40)

 50.0 (23.2)

 86.8 (32.7)

 59.3 (16.9)

 146.2 (55.1)

 21.7

 3 (120)

 50.4 (23.4)

 83.2 (31.3)

 59.6 (17.1)

 142.8 (53.7)

 19.5

 4 (360)

 50.2 (24.2)

 80.0 (31.0)

 56.2 (16.6)

 136.2 (52.8)

 17.6

 

Table 3: Summary of dam body weights

 Day post coitum

 Group 1 (0 mg/kg)

 Group 2 (40 mg/kg)

 Group 3 (120 mg/kg)

 Group 4 (360 mg/kg)

 0

 216.6

 215.2

 215.6

 207.5

 6

 265.6

 265.2

 266.0

 257.7

 16

 348.9

 352.0

 349.2

 337.7

 20

 405.0

 411.4

 408.8

 393.9

 

Table 4: Summary of reproduction data (dams with live foetuses)

 

 Group 1 (0 mg/kg)

 Group 2 (40 mg/kg)

 Group 3 (120 mg/kg)

 Group 4 (360 mg/kg)

 Number of dams

 23

 23

 23

 22

 Corpora lutea

 382

 385

 378

 381

 Mean

 16.6

 16.7

 16.4

 17.3

 S.D.

 1.8

 2.3

 2.3

 1.9

 Implantation sites

 329

 346

 335

 333

 % of corpora lutea

 86.1

 89.9

 88.6

 87.4

 Mean

 14.3

 15.0

 14.6

 15.1

 S.D.

 2.1

 2.0

 2.3

 1.6

 Pre-implantation loss

 53

 39

 43

 48

 % of corpora lutea

 13.9

 10.1

 11.4

 12.6

 Post-implantation loss

 35

 17 #

 16 #

 19 #

 % of implantation sites

 10.6

 4.9

 4.8

 5.7

 Embryonic deaths: total

 35

 17 #

 14 ##

 19 #

 Embryonic resorptions

 34

 13

 11

 14

 % of implantation sites

 10.3

 3.8

 3.3

 4.2

 Mean

 1.5

 0.6

 0.5

 0.6

 S.D.

 1.6

 0.6

 0.8

 0.8

 Foetal resorptions

 1

 4

 3

 5

 % of implantation sites

 0.3

 1.2

 0.9

 1.5

 Mean

 0.0

 0.2

 0.1

 0.2

 S.D.

 0.2

 0.4

 0.3

 0.4

 Foetuses

 

 

 

 

 No. of dams

 23

 23

 23

 22

 Total foetuses

 294

 329 #

 321 ##

 314 #

 % of implantation sites

 89.4

 95.1

 95.8

 94.3

 Mean

 12.8

 14.3

 13.9

 14.3

 S.D.

 2.6

 2.1

 2.2

 1.9

 Live foetuses

 294

 329

 319

 314

 Dead foetuses

 0

 0

 2

 0

 Malformed foetuses

 1

 0

 2

 0

 % of foetuses

 0.3

 0.0

 0.6

 0.0

 Mean

 0.0

 0.0

 0.1

 0.0

 S.D.

 0.2

 0.0

 0.3

 0.0

 Sex of foetuses

 

 

 

 

 Total males

 132

 146

 154

 154

 % of foetuses

 44.9

 44.4

 48.0

 49.0

 Mean

 5.7

 6.3

 6.7

7.0

 S.D.

 2.1

 2.7

 2.2

 1.9

 Total females

 162

 183

 165

 160

 % of foetuses

 55.1

 55.6

 51.4

 51.0

Mean 

 7.0

 8.0

 7.2

 7.3

 S.D.

 1.5

 2.8

 1.8

 2.2

  Weights of live foetuses (litter basis)  

 

 

 

         

 Males and females

 

 

 

 

 N (litters)

 23

 23

 22

 22

 Mean

 4.1

 4.2

 4.6

 4.4

 S.D.

 0.7

 0.8

 0.9

 0.8

 Runts

 

 

 

 

 N (litters)

 3

 1

 2

 2

 Mean

 1.0

 1.0

 1.0

 1.0

 S.D.

 0.0

 0.0

 0.0

 0.0

 Males

 

 

 

 

 N (litters)

 23

 23

 23

 22

 Mean

 4.2

 4.3

 4.7

 4.5

 S.D.

 0.7

 0.8

 1.0

 0.8

 Runts

 

 

 

 

 N (litters)

 2

 0

 1

 0

 Mean

 1.0

 0.0

 1.0

 0.0

 S.D.

 0.0

 0.0

 0.0

 0.0

 Females

 

 

 

 

 N (litters)

 23

 23

 23

 22

 Mean

 4.1

 4.1

 4.5

 4.3

 S.D.

 0.7

 0.7

 0.9

 0.7

 Runts

 

 

 

 

 N (litters)

 1

 1

 1

 2

 Mean

 1.0

 1.0

 1.0

 1.0

 S.D.

 0.0

 0.0

 0.0

 0.0

 Weights of placenta (litter basis)

 

 

 

 

 Males and females

 

 

 

 

 N (litters)

 23

 23

 23

 22

 Mean

 0.6

 0.6

 0.6

 0.6

 S.D.

 0.1

 0.1

 0.1

 0.1

 Weights of uteri (dam basis)

 

 

 

 

 N (dams)

 23

 23

 23

 22

 Mean

 78.2

 89.2 *

 91.6 *

 90.7 *

 S.D.

 16.2

 11.7

 18.4

 14.1

# Fisher's Exact test (Bonferroni-Holm-corrected) significant at level 5% (#), 1% (##)

* Dunnett test based on pooled variance significant at level 1%

 

Conclusions:
Under the conditions of the test, the repeat oral administration of the test material to pregnant rats showed no symptoms of cumulative toxicity or any embryotoxic or teratogenic potential up to 360 mg/kg bw/day.
Executive summary:

The test material was tested at dose levels of 0 (group 1), 40 (group 2), 120 (group 3) and 360 (group 4) mg/kg bw. Each group consisted of at least 24 female rats (pregnant rats: 23 in groups 1 to 3 and 22 in group 4). The test material was administered orally by gavage daily from days 6 to 15 of gestation. A standard dose volume of 5 mL/kg bw was used. Control animals were dosed with the vehicle alone over the same period.

Clinical conditions and reactions to treatment were recorded at least once daily. Body weights were recorded for days 0, 6, 16 and 20 of gestation. All surviving females were sacrificed on day 20 of gestation and the foetuses were removed by caesarean section. At necropsy, the females were examined macroscopically and live foetuses were weighed, sexed and examined for visceral and skeletal abnormalities.

No mortality was observed at any of the dose levels tested. No compound-related symptoms were observed in all treatment groups. Maternal body weight gain was not affected by treatment. No treatment-related abnormalities were found at necroscopy of the females. All females had viable foetuses. Pre-implantation loss, post-implantation loss, mean numbers of resorptions, embryonic deaths and total foetuses were not affected by treatment.

Mean foetal placental and uterus weights were not affected by treatment; foetal sex ratio was comparable in all group. No treatment-related foetal abnormalities were found at necroscopy; there were no treatment-related effects in the reproduction data. The examined foetuses showed no treatment-related malformations. The number of skeletal variations showed no treatment-related deviations. The number of skeletal ossifications in both the test groups and the control group were considered to be similar. The number of visceral variations in both the test groups and the control group were considered to be similar.

The results of the study show that repeat oral administration of the test material (days 6 - 15 post coitum) to pregnant rats caused no symptoms of cumulative toxicity up to 360 mg/kg bw/day. The test material reveals no embryotoxic or teratogenic potential at dose levels up to 360 mg/kg bw/day.

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:
The key study is GLP compliant and has a Klimisch score of 2 on the basis that the test material is a read-across substance. The study is GLP compliant conducted to a standardised guideline. The study was performed on a suitable structural analogue. The overall quality of the database is therefore high.
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

In the key study, the test material was tested at dose levels of 0 (group 1), 40 (group 2), 120 (group 3) and 360 (group 4) mg/kg bw. Each group consisted of at least 24 female rats (pregnant rats: 23 in groups 1 to 3 and 22 in group 4). The test material was administered orally by gavage daily from days 6 to 15 of gestation. A standard dose volume of 5 mL/kg bw was used. Control animals were dosed with the vehicle alone over the same period.

Clinical conditions and reactions to treatment were recorded at least once daily. Body weights were recorded for days 0, 6, 16 and 20 of gestation. All surviving females were sacrificed on day 20 of gestation and the foetuses were removed by caesarean section. At necropsy, the females were examined macroscopically and live foetuses were weighed, sexed and examined for visceral and skeletal abnormalities.

No mortality was observed at any of the dose levels tested. No compound-related symptoms were observed in all treatment groups. Maternal body weight gain was not affected by treatment. No treatment-related abnormalities were found at necroscopy of the females. All females had viable foetuses. Pre-implantation loss, post-implantation loss, mean numbers of resorptions, embryonic deaths and total foetuses were not affected by treatment.

Mean foetal placental and uterus weights were not affected by treatment; foetal sex ratio was comparable in all group. No treatment-related foetal abnormalities were found at necroscopy; there were no treatment-related effects in the reproduction data. The examined foetuses showed no treatment-related malformations. The number of skeletal variations showed no treatment-related deviations. The number of skeletal ossifications in both the test groups and the control group were considered to be similar. The number of visceral variations in both the test groups and the control group were considered to be similar.

The results of the study show that repeat oral administration of the test material (days 6 - 15 post coitum) to pregnant rats caused no symptoms of cumulative toxicity up to 360 mg/kg bw/day. The test material reveals no embryotoxic or teratogenic potential at dose levels up to 360 mg/kg bw/day.

The study was performed in line with a standardised guideline and under GLP conditions and reported to a high standard. The study was performed on the read-across substance cyclohexyl salicylate, a structural analogue of the registered substance. A reliability score of 2 was therefore assigned in accordance with the principles for assessing data quality as described in Klimisch (1997).

The supporting study, dosed Wistar rats with 0, 50, 200 and 700 mg/kg bw/day in a repeated dose and reproductive/developmental toxicity screen. The test material induced mortality in the high dose group and bodyweight (and bodyweight change), food consumption, haematological, clinical chemistry, microscopic and macroscopic changes. Effects on fertility were noticed in the females (some of which were attributed to the testicular effects observed in the male animals included in the screening study). In the high dose group, none of the dams delivered any pups. The test material was found to have no effect on pup viability nor on pup bodyweight development during post-partum days 1-4 in the two treatment groups that produced litters, furthermore no gross external anomalies reported. Under the conditions of the test, the no-observed-adverse effect level (NOAEL) was considered to be 200 mg/kg/day for both toxicity and developmental parameters, and the no-observed effect level (NOEL) was established as 50 mg/kg/day. The study was performed in line with a standardised guideline and under GLP conditions and reported to a high standard. . A reliability score of 1 was therefore assigned in accordance with the principles for assessing data quality as described in Klimisch (1997).

Justification for selection of Effect on developmental toxicity: via oral route:

Both available studies were performed to a high standard, however due to the limited observations performed in a screening study, the pre-natal developmental toxicity study was therefore selected as the key study.

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008, the substance does not require classification for reproductive or developmental toxicity.