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)

Key study: OECD 422: NOAEL 200 mg/kg bw/day

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
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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
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 for cross-reference:
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.
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August 2012 to 16 April 2013
Reliability:
1 (reliable without restriction)
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.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst, The Netherlands
- Weight at study initiation: 291 - 367 g (males); 189 - 239 g (females)
- Housing: individually in Makrolon type-3 cages with wire mesh tops
- Diet: Pelleted standard Harlan Teklad 2914C ad libitum
- Water: tap water ad libitum
- Acclimatisation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30 - 70%
- Air changes: 10 - 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 6th September 2012: To: 15th October 2012
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was weighed into a glass beaker and the vehicle added. The mixtures were stirred using a magnetic stirrer and stored at room temperature.

VEHICLE
- Lot/batch no. (if required): BCBG8285V
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days maximum
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no (after two unsuccessful matings, the female was sacrificed and the reproductive organs examined histopathologically)
- After successful mating each pregnant female was caged (how): Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analysed by GC-FID.

Concentration, homogeneity and stability were determined in samples taken after experimental start (see Table 1 in 'Any other information on materials and methods incl. tables').

Concentration and homogeneity were further determined (see Table 2 in 'Any other information on materials and methods incl. tables').
Duration of treatment / exposure:
males: 4 weeks; females: 7 weeks
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control (Group 1)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
700 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected on the basis of a non-GLP range-finding study (see details in repeat dose enpoint for OECD 422 study)
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Observations: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behaviour were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly (males); pre-mating period days 1-8 and 8-13 (females)

WATER CONSUMPTION: No

OTHER: neurobehavioural examination, clinical chemistry, and haematology were all examined and reported in the corresponding repeated dose toxicity summary.
Sperm parameters (parental animals):
- Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. If test material-related morphologic changes were detected in organs of high-dose animals, the same organs from mid-dose and low-dose animals were examined to establish a no-effect level if possible. Microscopic examination of the reproductive organs of all infertile males was made.
Litter observations:
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible) 1 and 4 post-partum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving males were sacrificed after at least 28 days treatment with the test material (when the males were no longer needed for the assessment of reproductive effects)
- Maternal animals: All surviving females were sacrificed on day 4 post-partum.

GROSS NECROPSY / ORGAN WEIGHTS
- Gross necropsy consisted of the testes and epididymides of all parental males were weighed separately. In addition, the following organs were selected from five males and five females from each group and weighed: adrenals, brain, heart, kidneys, liver, thymus and spleen.

HISTOPATHOLOGY
The following tissues were examined histologically: prostrate, seminal vesicles with coagulating gland, testes and epididymides (males); ovaries 9females); gross lesions, brain, spinal cord, heart, thymus, thyroids and parathyroids, small and large intestines, stomach, liver, kidneys, adrenals, spleen, trachea and lungs, uterus, urinary bladder, lymph nodes, peripheral nerve and bone marrow (males and females).
Postmortem examinations (offspring):
Pups were observed for any gross anomalies.
Statistics:
The following statistical methods were used to analyse food consumption, body weights and reproduction data:
- Mean and standard deviations were calculated.
- The Dunnett-test based on pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied instead of the Dunnett test when the could not be assumed to follow a normal distribution.
- Fisher's exact test was applied if the variables could be dichotomised without loss of information.
Reproductive indices:
Corpora lutea count
Duration of gestation
Implantation rate and post-implantation loss
Litter size
Offspring viability indices:
Sex ratios
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One control male was found spontaneously dead on day five and one 200 mg/kg bw/day male was found spontaneously dead on day 11; both deaths were considered to be due to an incidental influx of dosing solution into the respiratory tract. Two 700 mg/kg bw/day males were found spontaneously dead on days five and six of the pre-mating period.

Two 200 mg/kg bw/day females were found spontaneously dead on days 23 and 24 of the gestation period; the death of one of them was considered to be due to incidental influx of dosing solution into the respiratory tract. Three 700 mg/kg bw/day females were found spontaneously dead on days five, six and seven of the pre-mating period, one female of this dose group was sacrificed in extremis on day two of the pre-mating period and another female of this group was sacrificed in extremis on day 18 of the gestation period.

Apart from the deaths considered to be due to incidental influx of dosing solution into the respiratory tract, the death of one 200 mg/kg bw/day female was considered to probably be pregnancy toxemia but this was not considered to be directly attributable to the test material. In the remainder of the animals, the worsened general condition which was caused by the effects on the forestomach and kidneys and the continual stressful condition attributable to treatment with the test material were considered to be the cause of death.

No clinical signs were noted in control and 50 mg/kg bw/day males during the pre-mating period. Slight breathing noises were noted in a few 200 and 700 mg/kg bw/day males on one or two days of the pre-mating period. During the mating period, slightly soft faeces were noted in a few control males on one day. Slight breathing noises were noted in a few 50 mg/kg bw/day males on two days and in some 200 and 700 mg/kg bw/day males on some days of the mating period.

No clinical signs were noted in control, 50 and 200 mg/kg bw/day females during the pre-mating period. Slight breathing noises were noted in 700 mg/kg bw/day females on some days of the pre-mating period. Prostration and hunched posture were noted in some 700 mg/kg bw/day females on single days. Additionally, slightly ruffled fur and visible slight weight loss were seen in 700 mg/kg bw/day females on some days, slight convulsion was noted in one female on on day and slightly decreased activity was noted in a few females on one day of the pre-mating period.

During the mating period, no clinical signs were noted in control, 50 and 200 mg/kg bw/day females. Slightly decreased activity, hunched posture, slightly ruffled fur and visible slightly to moderate weight loss were noted in one 700 mg/kg bw/day females on two days. Slight to moderate hair loss was noted in one 700 mg/kg bw/day female on the ventral thorax, the abdomen, the genital region as well as the left and right thigh. Slight breathing noises were also noted in one female during some days of this period. On one day of the mating period, slight salivation was noted in one female of this dose group.

No clinical signs were noted during weekly observations in males of all groups during the pre-mating and mating period. In one 700 mg/kg bw/day female, slightly decreased activity and hunched posture were noted during the pre-mating period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the males, no statistically significant differences of the mean bodyweights and the mean bodyweight gain of males treated with 50 mg/kg of the test material were noted when compared with the control males during the pre-mating and mating period of the study. The bodyweight gain of males treated with 200 mg/kg was decreased (p < 0.01) on day two. The mean bodyweight of males treated with 700 mg/kg was slightly decreased (p < 0.05 or p < 0.01) during days three to give and the mean bodyweight gain was slightly decreased (p<0.01) compared with controls from day two until day 14 of the pre-mating period. This was not noted during the mating period.

In the females, no test material related effects on the mean bodyweights of the females were observed when compared with the controls during the pre-mating, mating, gestation and lactation period.

The mean bodyweight gain of females treated with 700 mg/kg of the test material was slightly decreased compared to the controls during days three to seven (p < 0.05 or p < 0.01) and on days 12 to 14 (p < 0.05 pr p < 0.01) of the pre-mating period. In females treated with 200 mg/kg, a slight decrease (p < 0.05 or p < 0.01) when compared to controls from days 11 to 21 of the gestation period was observed.

The mean food consumption of 700 mg/kg bw/day males was slightly decreased (p<0.05) when compared to controls during week one and increased (p<0.01) during week two. The mean food consumption of males of the other treated groups showed no statistically significant changes. 700 mg/kg bw/day females showed a tendency of a slightly decreased mean absolute and relative food consumption during the pre-mating period. During the first week of the pre-mating period, this decrease of the mean absolute food consumption was statistically significant (p<0.01).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Degeneration of spermatocytes/spermatids and increased incidence and/or severity of Sertoli cell vacuolation were recorded in testes of group 4 (700 mg/kg). Increase in cellular debris in the tubules, decrease in elongate spermatids and/or loss of germ cells were frequently observed in the affected testes of males in group 4. No abnormalities were identified on the Spermatogonia. Continual stressful condition of the animals suggested by thymic atrophy and/or adrenocortical hypertrophy was expected mainly in animals of group 4 and might have contributed to the testicular lesions, although the possible effects on the spermocytes/spermatids could not be excluded. Degenerative testicular changes recorded in the 700 mg/kg/day, irrespective of its cause were considered to be responsible for female infertility.

Increased cellular debris in the duct lumen as well as oligospermia in epididymides was recorded in males of group 4 (700 mg/kg). These were considered to be secondary effects of the testicular lesions observed in this group. Reduced secretion was recorded in the prostrate glands, seminal vesicle and coagulating glands in males of group 4 as well. There were no indicators of cellular/tissue injuries in these organs, and therefore, these findings were considered to be secondary changes following the testicular lesions, or the poor condition of these animals.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating Performance and Fertility:
Eleven females mated in groups 1, 2 and 3 and 7 or group four were mated within the first or second mating period.

The median and mean pre-coital times were unaffected by treatment with the test material. Mean pre-coital times were 2.9, 2.3, 3.9* and 3.4 days in groups 1, 2, 3 and 4, respectively. The median pre-coital times were 3, 3, 3, and 4 days in order of ascending dose level. For the one female (700 mg/kg /day) that mated during the pre-coital time was 6 days, it is not determined whether the mating was initially overlooked.

The fertility index was 90.9, 81.8, 81.8 and 62.5 % in the 0, 50, 200 and 700 mg/kg/day groups respectively. In the 700 mg/kg/day, the conception rate was similar to the fertility index (71.4 %).

All pregnant females treated with 700 mg/kg/day did not deliver any offspring. The gestation index was 100, 100, 77.8 and 0 % in the 0, 50, 200 and 700 mg/kg/day groups respectively.

Corpora Lutea Count:
The mean number of corpora lutea per dam was similar in all groups, 14.8, 14.2 and 14.1 in the 0, 50 and 200 mg/kg/day groups, the mean number of corpora lutea in the high dose group could not be determined due to the dams not delivering a litter.

Duration of Gestation:
The mean duration of gestation was unaffected by exposure to the test material. Mean duration of gestation was 21.6, 21.4, 21.9 and not determined (no litter delivered) in order of ascending dose level.

Implantation Rate and Post-Implantation Loss:
The mean number of implantations was slightly decreased in females treated with 50 and 200 mg/kg/day, this did not reach statistical significance. The post-implantation loss was increased slightly at the 50 mg/kg/day group, and increased at the 200 mg/kg/day group which reached statistical significance (p < 0.01). This was considered to be test material related.

The mean number of implantation per litter were 14.2, 13.2 and 11.7 at 0, 50 and 200 mg/kg/day. These were all within the historical control range. The number of implantations in the high dose group could not be determined as no litter was delivered.

Litter Size at First Litter Check:
In the 0, 50 and 200 mg/kg/day group, the mean birth indices were slightly decreased in a dose-response pattern (91.5, 84.0 and 61.0 (p < 0.01)). As none of the high dose group females delivered, this could not be calculated for this dose level. This effect was considered to be related to test material administration.

Postnatal Loss Days 0 - 4 Post Partum:
No effects on the postnatal loss between day 0 and 4 post partum were noted in females treated with 50 mg/kg/day or 200 mg/kg/day of the test item. The mean total postnatal loss was 1, 2, 1 and not detectable in groups 1, 2, 3 and 4 respectively.
The viability index was 99.2, 98.0% in groups 1 to 3 and it was not detectable in group 4 due to no delivery.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No statistically significant changes in the mean organ weights, organ to body weight ratios and organ to brain ratios were noted in females when compared with the control animals.

In males, the mean liver weight, the mean liver to body weight and the mean liver to brain weight ratio were increased (p < 0.01) in animals treated with 700 mg/kg/day of the test item when compared with controls. The mean thymus weight, the mean thymus to body weight ratio and the mean thymus to brain weight ratio were decreased (p<0.01) in males of this dose group when compared with controls. Both findings were considered to be test item related.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no gross lesions in the animals that died as a results of treatment with the test material, and the gross changes were considered to be incidental lesions, non-specific changes that are commonly observed in the decedent the lesions are considered to be related to stressful conditions and also spontaneous lesions which are associated with animals of the strain and age used in the study.

Incomplete collapse of the lung was observed in two animals (11 a control male and 31 a male from the 300 mg/kg group). This correlated with observed alveolar oedema (minimal in animal 11 and moderate in animal 31).

In animal number 31, slight mucosal necrosis was observed microscopically in the trachea. These microscopic changes were considered to be due to incidental influx (e.g. regurgitation) of the dosing solution into the respiratory tract, this was considered to be related to the morbidity of this animal.
In animal number 11, incidental influx of the dosing solution into the respiratory tract was suspected to be the cause of the animals morbidity, although histological changes could not be identified on the trachea die to progressive of autolysis.

Discolouration recorded in lungs, thymus and lymph node, which were correlated microscopically with congestion and/or agonal haemorrhage were considered to be non-specific changes commonly observed in the decedent.

The black-foci of the gastric fundus and the reduced splenic size, which were correlated microscopically with glandular stomach erosion and splicing follicular atrophy/reduced red pulp area respectively, were considered to be non-specific, secondary changes associated with a stressful condition.
Liquid and/or brown contents in the stomach and/or intestines were recorded in animals 40, 42 and 78 (2 males from the 700 mg/kg group and one female from the 700 mg/kg group). Such microscopic findings may be observed when there were haemorrhagic lesions in the alimentary tracts. In animal 78 (the female from the 700 mg/kg group), black-contents in the ileum and cecum were recorded. Although there were no histological abnormalities in these organs, glandular stomach erosion with mucosal regeneration was observed microscopically, and therefore, macroscopic findings in the ileum and cecum were considered to be associated with gastric injuries.

In the other two males from this group, microscopic changes that were considered to be attributable to macroscopic findings could not be identified in these animals due to the progression of autolysis.

Splenic constriction and dark red discolouration of the ovary were correlated with focal fibrosis and congestion respectively when examined microscopically. These were considered to be spontaneous changes which may be observed in rats of this strain and age.

Gross lesions in the survivors attributable to treatment with the test item were recorded in the liver and the thymus.

Enlarged liver was recorded in two males of group 1, one male of group 2 and five males of group 4, the incidence was higher in group 4 males.
Reduced thymic size was observed in four males of group 4.

All other gross lesions recorded in the animals surviving to termination were within the normal background alterations for this age and strain of rat.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The test material induced histomorphological changes in testes, epididymides, prostate glands, seminal vesicles, coagulating glands, kidneys, stomach, liver, adrenal glands, thymus and trachea.

In the females, no specific effects were observed in the ovaries.

In the kidney, increased incidence and/or severity of tubular degeneration/regeneration were recorded in both sexes of group 4 (700 mg/kg), these degenerative changes were considered to be adverse.

In the stomach, hyperkeratosis and/or squamous cell hypertrophy (increased mucosal thickness) of the forestomach were recorded in animals of groups 3 and 4 (200 and 700 mg/kg, respectively). In addition to these findings, squamous cell hyperplasia and ulcer were recorded in the forestomach of the animals that died in group 4. These findings were considered to be attributable to the irritating properties of the test material. Hyperkeratosis and squamous cell hypertrophy of the forestomach were considered to be a simple reactive change to the irritative stimuli of the test material and was therefore not considered to be an adverse effect. The observed ulceration and squamous cell hyperplasia were tissue injury and its reparative process and was therefore considered to be adverse.

In the liver, centrilobular hepatocellular hypertrophy was recorded in both sexes of groups 3 and 4 (200 and 700 mg/kg, respectively). This was considered to be related to metabolism and adaptive in character, and there were no further indications of liver injuries. This was therefore not considered to be adverse.

Epithelial hypertrophy and regenerated mucosal epithelium of trachea were sporadically recorded in the survivors of groups 2 and 4. Mucosal necrosis and regenerated mucosal epithelium were also recorded in the decedent of groups 3 and 4. In addition, a slight increase in the group mean severity of mucosal/submucosal inflammatory cell infiltration was recorded in group 4. These were not caused by the systemic toxicity of the test material and were considered to be related to incidental influx (e.g. regurgitation) of dosing solutions containing the test material into the respiratory tract. The irritating properties of the test material was attributed to the slightly higher incidence in severity of the mucosal injuries in the groups treated with the test material compared to the control group.

OTHER FINDINGS (PARENTAL ANIMALS)
Neurobehavioral examination: None of the parameters investigated during the functional observational battery gave an indication of a test material-related effect.

Haematology: No test material related changes in the investigated haematology parameters were noted in males and females when compared with the control animals.

Clinical chemistry: Mean bilirubin was decreased in males treated with 200 and700 mg/kg/day which demonstrated a dose response relationship. Mean triglyceride value of males treated with 700 mg/kg/day was increased. The mean content of albumin was increased, the mean content of globulin was decreased and (mean albumin to globulin ratio was therefore increased) in males treated with 700 mg/kg/day.

The mean values of bilirubin were decreased in females treated with 200 and 700 mg/kg/day.

Mean triglyceride value of females treated with 700 mg/kg/day.

The mean value of protein was decreased in females treated with 200 and 700 mg/kg/day. The mean content of globulin was decreased in females treated with the test material the resulting mean albumin to globulin ratio was increased in females treated with 700 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive effects and parental toxicity
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at this dose
Critical effects observed:
no
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Litter Data - F1 pups:
External Examination at First Litter Check and during Lactation:
No test material related findings were noted in the first litter check or during the first four days post-partum.

Sex Ratios:
The mean number of pups at the first litter check was decreased in a dose-response pattern. No pups were delivered by females in the high dose group. The ratio of males to females was 50/50 in the 0 mg/kg/day group and was 45/55 and 36/64 in the 50 and 200 mg/kg/day group. A test material effect could not be discounted.

Body Weights to Day 4 Post Partum:
Mean pup weights on day 1 and 4 post-partum were unaffected by maternal treatment. The mean bodyweights of pups in the 50 mg/kg/day group was slightly increased (p < 0.05), however, as it was only seen at this dose level, it was considered incidental.

No test material related macroscopic findings were observed in the pups at necropsy.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see discussions
Critical effects observed:
no
Reproductive effects observed:
not specified

Table 3: Incidence and Severity (mean) of main findings in testes and epididymides

Incidence/Mean Severity

Group 1

Group 2

Group 3

Group 4

Testes

11 M

7 M

7 M

11 M

Degeneration, spermatocytes/spermatids

0

0

0

10/1.5

Sertoli cell vacuolation

2/1.0

1/1.0

0

9/1.8

Epididymides

11 M

7 M

7 M

11 M

Increased cellular debris in the lumen, with/without decreased normal maturing spermatazoa

0

0

1/1.0

9/1.2

Oligospermia

0

0

0

2/4.0

 

Table 4: Sperm staging

Group

Animal No.

Stage

Cell Population

Findings

1

1

Complete

Complete

Sertoli cell vacuolation, unilateral, minimal

2

Complete

Complete

None

3

Complete

Complete

None

4

Complete

Complete

None

5

Complete

Complete

None

6

Complete

Complete

None

7

Complete

Complete

Sertoli cell vacuolation, unilateral, minimal

8

Complete

Complete

None

9

Complete

Complete

Vasculitis, unilateral, minimal

10

Complete

Complete

None

11

Complete#

Complete#

None

2

12

Complete

Complete

Tubular atrophy, focal, unilateral, minimal;

Sertoli cell vacuolation, unilateral, minimal

13

Complete

Complete

None

14

Complete

Complete

None

15

Complete

Complete

None

16

Complete

Complete

None

17

Complete

Complete

None

21

Complete

Complete

None

3

23

Complete

Complete

Vasculitis, unilateral, minimal

24

Complete

Complete

None

25

Complete

Complete

None

26

Complete

Complete

None

27

Complete

Complete

None

28

Complete

Complete

Tubular atrophy, focal, unilateral, slight

31

Complete

Complete

None

4

34

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, slight (with increase in cellular debris and decrease in elongated spermatids in tubules, and loss of germ cells);

Sertoli cell vacuolation, bilateral, moderate

35

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, slight (with increase in cellular debris and decrease in elongated spermatids in tubules);

Sertoli cell vacuolation, bilateral, slight

36

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, slight (with increase in cellular debris and decrease in elongated spermatids in tubules, and loss of germ cells);

Sertoli cell vacuolation, bilateral, slight

37

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral minimal (with increase in cellular debris in tubules);

Sertoli cell vacuolation, unilateral, minimal

38

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, slight (with increase in cellular debris and decrease in elongated spermatids in tubules, and loss of germ cells);

Sertoli cell vacuolation, bilateral, slight

39

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, minimal (with increase in cellular debris in tubules);

Sertoli cell vacuolation, bilateral, minimal

40

Complete#

Complete#

Degeneration, spermatocytes/spermatids, bilateral minimal

41

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, minimal (with increase in cellular debris in tubules);

Sertoli cell vacuolation, bilateral, slight

42

Complete#

Complete#

None

43

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, minimal (with increase in cellular debris in tubules);

Sertoli cell vacuolation, bilateral, minimal

44

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, minimal (with increase in cellular debris in tubules);

Sertoli cell vacoulation, bilateral, slight

# Autolysis had begun, but the representative stages/cell populations were identified and the completeness confirmed

 

Table 5: Incidence and Severity (mean) of main findings in prostate, and coagulating glands and seminal vesicles

Incidence/Mean Severity

Group 1 (n = 10)

Group 2 (n = 7)

Group 3 (n = 6_

Group 4 (n = 9)

Prostate gland - Reduced secretion

0

0

0

3/1.0

Seminal vesicles - Reduced secretion

0

0

0

2/1.0

Coagulating glands - Reduced secretion

0

0

0

2/1.0

 

Table 6: Summary of Reproductive Performance

Dose (mg/kg/day)

0

50

200

700

Number of females paired

11

11

11

8

Number of females mated

11

11

11

7

Number of unscheduled deaths

0

0

2

5

Number of females not pregnant

0

0

2

3

Number of females pregnant but did not deliver

0

0

2

5

Number of females which reared their pups until day 4 post-partum

10

9

7

0

 

Conclusions:
Under the conditions of the test, the no-observed-adverse effect level (NOAEL) for reproductive effects and parental toxicity was considered to be 200 mg/kg/day for males and females, and the no-observed effect level (NOEL) was established as 50 mg/kg/day in both sexes.
Executive summary:

The test material was administered to male rats for at east 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. The test material was applied at 0, 50, 200 and 700 mg/kg bw/day (groups 1 to 4, respectively). The following effects were recorded:

One male in group one, one male and two females of group three and two males and five females of group four died or were killed in extremis prematurely. The cause of death of the male in group one and one male and one female in group three was considered to be incidental influx of the dosing solution into the respiratory tract. In the other animals, the stressful condition attributable to treatment of the test material was considered to be the cause of death.

None of the parameters investigated during the functional observational battery indicated a test material-related effect.

The mean food consumption of 50 and 200 mg/kg bw/day males were not statistically significant when compared to the controls. The mean food consumption of 700 mg/kg males was slightly decreased in week one and slightly increased in week two when compared to the controls. 700 mg/kg bw/day females showed a tendency of a slightly decreased mean absolute and relative food consumption on the pre-pairing period. During the first week of this period, the decreased of the mean absolute food consumption was statistically significant. In 200 mg/kg bw/day females, the mean food consumption was slightly decreased during the lactation period.

No statistically significant differences in mean body weights and mean body weight gain of 50 mg/kg bw/day males were noted compared to the controls during the pre-pairing and pairing period. The mean body weight of 700 mg/kg bw/day males was slightly decreased during days three to five and the mean body weight gain was slightly decreased when compared to the controls from day two to day 14 of the pre-pairing period (this was not noted during the pairing period). No effects on mean body weights of females were observed during the pre-pairing, pairing, gestation and lactation periods. The mean body weight gain of 700 mg/kg bw/day females was slightly decreased compared to controls during days three to seven and on days 12 to 14 of the pre-pairing period. The mean body weight gain was slightly decreased in 200 mg/kg bw/day females when compared to controls from day 11 to 21 of the gestation period.

No changes in haematology parameters were noted in males and females when compared to the controls.

The mean values of bilirubin were decreased in 200 and 700 mg/kg bw/day males when compared to the controls; this decrease showed a dose-response relationship and is therefore considered to be test material related. The mean triglyceride value of 700 mg/kg bw/day males was increased when compared to the controls; this value exceeds the mean standard deviation of the historical reference data and is therefore considered to be test material related. The mean albumin content was increased, the mean globulin content was decreased and, due to this, the mean albumin to globulin ratio was increased in 700 mg/kg bw/day males; it cannot be excluded that this is test material related. The mean bilirubin values decreased in 200 and 700 mg/kg bw/day females when compared to the controls; this decrease showed a dose response relationship and is therefore considered to be test material related. The mean triglyceride value in 700 mg/kg bw/day females was increased when compared to the controls; this value exceeds the mean standard deviation of the historical reference data and is considered to be test material related. The mean protein value decreased in 200 and 700 mg/kg bw/day females when compared to the controls. The mean content of globulin decreased in all treated groups compared to the controls; due to this the mean albumin to globulin ratio increased in 700 mg/kg bw/day females when compared to the controls and it cannot be excluded that this is test material related.

No statistically significant differences in mean organ weights, mean organ to body weight ratios and mean organ to brain weight ratios were noted in treated females when compared to controls. In 700 mg/kg bw/day males, the mean liver weight, the mean liver to body weight ratio and the mean liver to brain weight ratio were statistically significantly increased when compared to controls. The mean thymus weight, the mean thymus to body weight ratio and the mean thymus to brain weight ratio of this dose group were statistically significantly decreased when compared to controls.

In the kidney, increased incidence and/or severity of tubular degeneration/regeneration were recorded in both sexes at 700 mg/kg bw/day and such degenerative lesions were considered to be adverse. In the stomach, hyperkatosis and/or squamous cell hypertrophy were recorded in 200 and 700 mg/kg bw/day animals. In addition to these findings, squamous cell hyperplasia and ulcer were recorded in the forestomach of 700 mg/kg bw/day animals. These findings were considered to be related to the irritant property of the test material.

In the liver, centrilobular hepatocellular hypertrophy was recorded in both sexes of 200 and 700 mg/kg bw/day animals. This was considered to be of metabolic nature and adaptive character and there were no further indicators of liver injuries. Epithelia hypertrophy and regenerated mucosal epithelium of trachea were sporadically recorded in 50 and 700 mg/kg bw/day survivors. Mucosal necrosis and regenerated mucosal epithelium were also recorded in the decedent of 200 and 700 mg/kg bw/day animals. In addition, a slight increase in the group mean severity of mucosal/submucosal inflammatory cell infiltration was recorded in 700 mg/kg bw/day animals. These were considered to be associated with incidental influx of the dosing solution containing the test material.

Degeneration of spermatocytes/spermatids and increased incidence and/or severity of Seroli cell vacuolation were recorded in testes of 700 mg/kg bw/day males. Increase in cellular debris in the tubules, decrease in elongate spermatids and/or loss of germ cells were frequently observed in the affected males in this group. No abnormalities were identified in spermatogonia. Continual stressful condition being suggested by thymic atrophy and/or adrenocortical hypertrophy was expected mainly at 700 mg/kg bw/day and might have contributed to the testicular lesions although the possible direct effects of the test material on spermatocytes/spermatids could not be excluded. Degenerative testicular changes recorded in group four animals, irrespective of the cause, were considered to be responsible for female infertility and considered to be adverse. Increased cellular debris in the duct lumen as well as oligospermia in epididymides was recorded in 700 mg/kg bw/day males. These were considered to have been secondary events following testicular lesions. Reduced secretion was recorded in the prostate glands, seminal vesicles and coagulating glands in 700 mg/kg bw/day males. There were no indicators of cellular/tissue injuries in these organs and, therefore, these findings were considered to be secondary changes following the testicular lesions caused by worsened general condition.

The mean precoital time was not affected by the treatment with the test material at any dose level. The fertility index was slightly decreased in animals treated with 700 mg/kg/day in comparison to controls. The post-implantation loss was slightly increased in females treated with 50 mg/kg/day and in females treated with 200 mg/kg/day of the test material when compared with controls. In the 0, 50 and 200 mg/kg/day groups, the mean birth indices were slightly decreased in a dose response pattern. The indices were 91.5, 84.0, 61.0 and the index was not detectable in group 4 due to no delivery. The mean litter size at first litter check was also decreased with increasing doses 13.0, 11.1, 7.1 and not detectable in group 4 due to no delivery. These findings were considered to be test material related.

 

The mean number of pups at first litter observation was decreased in a dose-response. No pups were delivered by females treated with 700 mg/kg/day. The ratio of males to females was 50/50 in the control group this appeared to be skewed by test material administration to to 45/55 in the group treated with 50 mg/kg/day to 36/64 in the group treated with 200 mg/kg/day. It could be not excluded that this shift was test material related. No test material-related differences in the mean body weight development of pups were noted in the 50 mg/kg/day or 200 mg/kg/day groups when compared to the controls. At necropsy, there were no abnormal findings in the pups.

 

Under the conditions of the test, the no-observed-adverse effect level (NOAEL) for reproductive effects and parental toxicity was considered to be 200 mg/kg/day for males and females, and the no-observed effect level (NOEL) was established as 50 mg/kg/day in both sexes.

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 OECD 422 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

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

Current study: OECD 422: NOAEL 200 mg/kg bw/day

Pre-natal devlopmental toxicity study (OECD 414) study on the substance in progress on will become the key study once available.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting 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
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
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 or test system 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 OECD 422 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 classification or non-classification

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

Additional information