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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23. May 2003 - 05. Aug. 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
test item : Benzoic acid isononylester
ID no. : 0649/82188
Batch no. : 1276/00576
Appearance : colourless, clear liquid
Storage conditions : room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: (P) Males: 185.9 - 201.0 g; Females: 160.8 - 182.3 g
- Fasting period before study: no
- Housing: The animals were housed in a limited access rodent facility. During the pre-mating period, animals were housed up to 5 of one sex to a cage, in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent paper which was inspected and changed at least 3 times a week. During the mating period, animals were housed on the basis of one male to one female in clear polycarbonate cages measuring 42x26x18cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily. The males were re-caged after mating as they were be fore mating. After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring 42x26x18cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Suitable nesting material was provided and was changed at least 3 times a week.
- Diet: A commercially available laboratory rodent diet (Altromin MT pelleted diet, D-32770 Lage, Postfach 1120, Germany) was offered ad libitum throughout the study.
- Water: ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: prepared daily in polyethylene glycol (PEG 400)
VEHICLE
- Justification for use and choice of vehicle (if other than water): standard vehicle in toxicology studies
- Concentration in vehicle: 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of mating until sperm identification and/or copulation plugs were found. The pairing combinations of any animals which have not had positive identification of mating after 14 days of pairing will be changed within each treatment group. The subsequent pairing will be monitored for mating as described above for a maximum period of 14 days. If no mating oceurs the females will be killed at least 7days after the last day of the mating session.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory . Sampies of the formulations prepared during the first and the last week of treatment were analysed to check the concentration and the homogeneity.
Duration of treatment / exposure:
Males: Animals were dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks prior to pairing and thereafter through the day be fore necropsy. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females: Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during gestation and post-partum periods, until the day before necropsy (Day 4 post-partum). During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post-coitum and on Day 1 post-partum. Thereafter individual dose volumes remained constant.
Frequency of treatment:
once daily
Details on study schedule:
On the day of allocation, 7 days prior to the start of treatment, all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The rats were allocated to the 4 groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Males: Animals were dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks prior to pairing and thereafter through the day be fore necropsy. Females: Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during gestation and post-partum periods, until the day before necropsy (Day 4 post-partum).
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Details on study design:
- Dose selection rationale: Dose selection on basis of a confirmatory study (RTC 40330EXT)
- Rationale for animal assignment (if not random): random
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day on working days, and once on weekends and public holidays. Examination of individual animals for signs of reaction to treatment was carried out daily, prior to dosing, 30 minutes, 1 and 2 hours after dosing.
- Full records were maintained for all measurements and observations.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once per treatment week, and whenever possible at 7 day intervals, each animal was observed and any clinical signs were recorded.


BODY WEIGHT: Yes
- Time schedule for examinations: Males: Animals were weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy. Females: Animals were weighed on the day of allocation to treatment groups, weekly from the first day of treatment to mating, on Days 0, 7, 14 and 20 post-coitum and on Days 1 and 4 postpartum.


OTHER:
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
As soon as possible after parturition (Day 0 or 1 post-partum), the total litter size (live and dead) was counted, live pups were individually identifIed within the litter, sexed and examined for external abnormalities. All litters were weighed on Day 1 post-partum. All litters were examined daily for dead and abnormal pups. The pups were also weighed on Day 4 post-partum. All pups found dead were necropsied.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: The males were killed after the mating of females with carbon dioxide.
- Maternal animals:The females with live pups were killed on Day 4 post-partum with carbon dioxide. Females which had not given birth 25 days after the positive identification of mating were killed shortly after.


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


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues of abnormalities, seminal veshicles. epididymides, testes, prostate gland, ovaries, uterus and vagina were preserved. Detailed histopathological examination was performed on ovaries, testes, and epididymides from the control and the high dose groups.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Necropsy of pups also comprised the confirmation of sex by gonadal inspection.


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
SACRIFICE
- All viable pups were euthanised by intrascapular injection of Tanax on Day 4 post-partum.


GROSS NECROPSY
All pups found dead in the cage were necropsied. All live pups were killed on Day 4 postpartum and examined for the following:
a) external and internal abnormalities;
b) sex confirmation by gonadal inspection.
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett's test or a modified t test, depending on the homogeneity of data. The homogeneity of the data was assessed by Bartlett's test. Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test. The mean values, standard deviations were calculated from actual values in the computer without rounding off. Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Details on results (P0)

Fate of females: Two unscheduled deaths occurred during the study: one female in group 2 (animal no. 15090033) was sacrificed for humane reasonson Day 9 post-coitum and another female of the same group (animal no. 15090021) was found dead on Day 0 post-partum. A total of 14 live pups from this latter dam were humanely sacrificed. A total of 10 females were found not pregnant at necropsy, 4 in the control group, 1 in group 2 which wassacrificed for humane reasons on Day 9 post-coitum, 2 in group 3 and 3 in group 4. One female in group 4 (animal no. 15090065) had total litter losson Day 0 postpartum. The number of females with live young at Day 4 post-partum was 6 to 8.
Clinical signs and pre- and post-dose observations: Clinical signs observed at weekly intervals in FO males and females were limited to common
conditions of the skin and fur. Rales were occasionaIly observed at pre- and post-dose observations, in controls and in groups 2 and 4 male animals, and in groups 3 and 4 females (data not tabulated). Soft faeces were recorded during cages observation for control and treated groups.
Body weights: A statistical significant lower body weight was observed in male animals of group 2 compared to controls on study day 15 (dosing phase). This difference was not considered treatment related since no other differences in body weight and/or body weight gain were noted between the treated and control groups. Body weight and body weight gain for female animals were generaIly comparable between the treated and control groups before mating and during gestation and post-partum periods.
Food consumption: Food consumption was unaffected by the treatment.
Reproductive parameters: Irregular oestrus cycles were observed in treated groups and also in controls. However, a high incidence of animals with irregular oestrus cycles was noted in the treated groups compared to controls. This finding was not considered to be of toxicological significance
since no effects on relevant fertility parameters were observed. The copulatory and fertility index as weIl as the pre-coital intervals were not affected by the treatment.
Implantation, pre-birth loss data and gestation length: The gestation periods were similar between the groups. No significant differences were observed for the number of implantations, corpora lutea and pre-birth loss in treated groups compared to controls.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: overall results; body weight, body weight gain, food consumption, fertility index, pre-coital interval, copulatory index, histopathology of ovaries and testes, litter development
Remarks on result:
other: Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

Litter data: The mean litter and pup weights were sligthly reduced in group 4 compared to controls at birth as weIl as on Day 4 post-partum.
Sex ratios: No statistically significant differences were observed in the sex ratios between the groups.
Pre-weaning clinical signs of Fl pups: Pre-weaning clinical signs did not show treatment-related effects.
Necropsy findings in decedent pups: Decedent pups were found in treated groups including controls. One female in group 2 was found dead on Day 0 post-partum and a total of 14 pups were humanely sacrificed. No abnormalities were noted at necropsy for these pups (data not presented). One female in group 4 had total litter loss. No relevant fmdings that could be treatment-related were reported.
Necropsy findings in Fl pups at Day 4 post-partum: No significant fmdings were observed between the groups at necropsy of the pups.
Absolute and relative organ weights in FO males: Statistically significant higher relative organ weights of epididymides and testes were observed in group 2 compared to controls. These differences were not considered to be of toxicological relevance since no dose-dependency was observed.
Macroscopic observations: No macroscopic observation was reported in the treated animals that could be considered to be treatment-related.
Microscopic observations: The histopathological examination of the ovaries, testes and epididymides did not reveal any evident differences between the findings observed in the treated and control animals that could be considered treatment-related. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No abnonnalities were noted.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Litter data at birth and on Day 4 post-partum - Group mean data

 

 

At birth

On Day 4

Group

 

Litter total

Live litter size

Pup loss %

Litter wt (g)

Mean pup wt (g)

Live litter size

Cumulative loss %

Litter wt. (g)

Mean pup wt. (g)

1

(n)

6

6

6

6

6

6

6

6

6

 

Mean

14.7

14.2

3.5

86.6

6.4

13.3

9.3

128.6

9.6

 

SD

1.2

1.5

3.9

16.5

0.5

1.6

4.3

28.4

1.1

 

 

 

 

 

 

 

 

 

 

 

2

(n)

9

9

9

8

8

8

8

8

8

 

Mean

14.3

13.9

3.8

87.4

6.5

13.3

8.8

122.9

9.4

 

SD

2.7

3.1

7.9

18.9

0.5

3.3

8.4

26.2

1.2

 

 

 

 

 

 

 

 

 

 

 

3

(n)

8

8

8

8

8

8

8

8

8

 

Mean

14.0

13.4

4.0

82.3

6.4

13.0

6.3

120.0

9.5

 

SD

3.9

3.7

7.0

17.5

0.8

3.6

9.3

23.6

1.4

 

 

 

 

 

 

 

 

 

 

 

4

(n)

7

7

7

6

6

6

6

6

6

 

Mean

14.6

11.3

22.2

69.6

5.9

11.7

19.1

100.6

8.8

 

SD

1.8

5.7

35.6

12.4

0.6

2.7

12.0

12.8

1.2

 

 

 

 

 

 

 

 

 

 

 

* = Statistically significantly different from control group value at p < 0.05

Applicant's summary and conclusion

Conclusions:
On the basis of the results, the dosage of 1000 mg/kg/day could be considered as the NOAEL.
Executive summary:

The effects on reproduction and development of the offspring were evaluated after oral administration of BENZOIC ACID ISONONYLESTER in male and female rats. The test item was given before and during mating and throughout the gestation period until Day 3 post-partum at dosages of 100, 300 and 1000 mglkg/day. Body weight, body weight gain and food consumption were not affected by treatment in male or female animals. Reproductive parameters such as fertility index, pre-coital interval and copulatory index did not show significant differences in treated groups compared to controls. A slight reduction in mean pup weight and consequently in litter weight was noted in group 4 females at birth and on Day 4 post-partum. The histopathological examination of the ovaries, testes (including the stage in the spermatogenic cycle) did not reveal any evident differences between the findings observed in the treated and control animals. On the basis of the results, the dosage of 1000 mg/kg/day could be considered as the NOAEL.