Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): SAT 940037 (cyclohexylsalicylat)
- Physical state: colourless liquid
- Storage condition of test material: at ambient temperature
- Stability under test conditions: The test material was demonstrated to be stable in the vehicle (corn oil) for at least 72 hours.
- Molecular formula (if other than submission substance): C13H16O3
- Molecular weight (if other than submission substance): 220.27
- Smiles notation (if other than submission substance): C(=O)(c1c(O)cccc1)OC1CCCCC1

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland GmbH, Niederlassung Sulzfeld, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: (P) males were approximately 6 wks, females were approximately 8 weeks.
- Weight at study initiation: (P) Males: 172-262 g; Females: 178-221 g
- Housing: singly in Makrolon cages
- Diet: 'Ssniff R-Z' pelleted diet ad libitum
- Water: tap water ad libitum
- Acclimation period: 13 days (males), 12 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature: 21.5 +/- 1.5°C
- Humidity: 40-70%
- Air changes: 16 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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