Benzoic acid, 4-[(1-oxodecyl)oxy]-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP and OECD Guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (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

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: Groups 1 to 4: 12 males and 12 females per group. Group 10 (reserve) 2 males and 2 females
- Total Number of Animals: 50 males and 50 females
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 249 to 390 g (males) and 205 to 229 g (females)
- Identification: Cage card and individual animal number (ear tattoo). On day 1 post partum, pups were individually tattooed with Indian ink.
- Randomization: Randomly allocated to groups by body weight.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temperature range: 20.5 - 23 °C; relative humidity range in general between 35% and 70% ). The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
- Accommodation: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 43/12) rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of respective samples were performed.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

DOSE FORMULATIONS

The dose formulations were not corrected for purity and were prepared as supplied by the Sponsor.

The dose formulations were prepared weekly as indicated by the results of stability analyses in the study D49720.

DOBA was weighed into a glass beaker on a tared Mettler balance. The vehicle was added and the mixtures were stirred using a magnetic stirrer and/or ultraturrax and used at room temperature (15 - 25 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STABILITY AND STORAGE OF DOSE FORMULATIONS

- Stability of Dose Formulations: For up to one week, based upon the results of stability analyses performed during Harlan Laboratories study D49720.
- Storage of Dose Formulations: At room temperature (20 ± 5 °C) in glass beakers.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
- Frequency of Administration: Daily
- Daily Target Dose Level: 0 mg/kg/day (Group 1), 100 mg/kg/day (Group 2), 300 mg/kg/day (Group 3) and 1000 mg/kg/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rats.
- Dose Volume: 5 mL/kg
- Dose Concentrations: 0 mg/mL/day (Group 1), 20 mg/mL/day (Group 2), 60 mg/mL/day (Group 3) and 200 mg/mL/day (Group 4)

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

As female no. 80 did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was carried out.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD

The dose formulations were analyzed using a HPLC method provided by the Sponsor and adapted by Harlan Laboratories. The samples (approximately 1 g each) were delivered to the analytical laboratory.

Transport of Dose Formulations to Analytical Laboratory: At ambient temperature (20 ± 5 °C)
Storage of Dose Formulations in Analytical Laboratory: Frozen (-20 ± 5 °C)

Samples of dose formulations were not discarded without the written authorization of the study director. Solutions of samples were discarded latest 3 months after preparation without further notice.

The test item served as analytical standard/reference item.

Concentration and homogeneity of dose formulations were determined in samples taken as follows:

- Concentration and Homogeneity after Experimental Start (24-Jan-2013) and during Week 6 (28-Feb-2013): b (middle) in Group 1 and a (top), b (middle), c (bottom) in Groups 2 to 4.

RESULTS

The absence of the test item in the vehicle control samples (corn oil) was confirmed.

The DOBA concentrations in the dose formulations prepared for groups 2 - 4 ranged from 81.8% to 104.9% with reference to the nominal concentration. Thus, they were all within the accepted range of ±20% with reference to the nominal concentration.

The homogeneous distribution of DOBA in the preparations was approved because single results found did not deviate more than 3.7% from the corresponding mean and met the specified acceptance criterion of ≤15%.

In conclusion, the results indicate the accurate preparation of the test item DOBA in vehicle during this study.

Duration of treatment / exposure:

42 days (males) and approximately 7 weeks (females)

Frequency of treatment:

Once daily

Details on study schedule:

MALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum per pairing period*
- Treatment Ends: On day before sacrifice
- Necropsy: After 42 days of treatment

FEMALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum per pairing period*
- Gestation: Approximately 21 days
- Treatment Ends: On day 3 post partum; females not giving birth on day 24 post coitum
- Necropsy: Dams and pups on day 4 post partum; females not giving birth on day 25 post coitum


* One female (no. 80) did not mate during the 14-day pairing period. Therefore, a second pairing of this female with a male in the same group, which had already mated successfully, was carried out. Since there was a second pairing period for one female, the length of the pairing phase was 15 days for all males.

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

The aim of this study was to assess the possible effect of DOBA on male, female fertility and embryofetal development in Wistar rat. This study was also aimed to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The test item was administered once daily orally (by gavage) to male and female rats throughout the pre-pairing and pairing periods, after pairing (males), gestation and lactation periods (females) including the day before scheduled necropsy.

This study should provide information to assess the need to conduct further investigations and may provide guidance in the design of subsequent studies.

Positive control:

Not required

Parental animals: Observations and examinations:

VIABILITY/MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations (once daily, during acclimatization and up to the day of necropsy). Additionally, females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

- Males weekly during pre-pairing and after pairing periods
- Females during pre-Pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.

No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

HISTOPATHOLOGY: Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

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):

TERMINATION AND NECROPSY

Males were sacrificed after 42 days of treatment. Dams were sacrificed on day 4 post partum. When birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes. Special attention was directed at the organs of the reproductive system.

Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS

At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.

TISSUE PRESERVATION

The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution.


HISTOTECHNIQUE

All preserved organ and tissue samples were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.

HISTOPATHOLOGY

Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals which died spontaneously or had to be terminated in extremis.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on any females that did not give birth.

A histopathology phase report was provided by the principal investigator. A histopathology peer review was performed by W. Henderson.

Postmortem examinations (offspring):

Pups were sacrificed on day 4 post partum. At the scheduled sacrifice, all pups were weighed and sacrificed by an injection of sodium pentobarbital. All pups were examined macroscopically for any structural changes. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

Statistics:

The following statistical methods were used to analyze food consumption, body weights, organ weights, macroscopic findings, and reproduction data:

- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a 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 (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated: mean precoital time, percentage mating, fertility index, conception rate, post-implantation loss, gestation index, birth index and viability index.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex ratio and viability index.

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:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

1. IN-LIFE DATA

VIABILITY / MORTALITY

There was no test item-related effect on viability/mortality.

Female no. 51 of the control group died after an accident during application on day 12 of the pre-pairing period.

Male no. 26 (300 mg/kg/day) was killed in extremis on day 5 after pairing because of swelling in the neck region that made a correct test item-administration impossible. At necropsy, a lesion at the brain (cavity with watery fluid) was noted.

At 1000 mg/kg/day, female nos. 89 and 94 died prematurely on days 14 and 17, respectively, of the gestation period (both on 24-Feb-2013). Since both animals died on the same day and shortly after dosing, a correlation of these deaths to the gavage procedure seems probable, although no corresponding macroscopic findings were observed at necropsy (see Section 4.4.2). Therefore, the deaths of these animals are considered to be not related to the test item.

CLINICAL SIGNS OR OBSERVATIONS

No test item-related clinical signs were observed.

A kinked tail apex was recorded for male no. 27 (300 mg/kg/day) from day 10 of the pre-pairing period onwards. In male no. 26 (300 mg/kg/day), swelling in the neck region and slightly tilted head was noted during the after pairing phase. The animal was subsequently killed for ethical reasons (see above).

No further clinical signs were noted in males or females at any dose level.

FOOD CONSUMPTION OF MALES

No test item-related effect on food consumption was noted in males.

The overall differences in mean food consumption between control group and males treated with 100, 300 and 1000 mg/kg/day, respectively, were 1.8%, -5.0% and +0.9% during the pre-paring phase, and +4.5%, +2.5% and +1.0%, during the after-pairing phase.

FOOD CONSUMPTION OF FEMALES

No test item-related effect on food consumption was noted in females.

Slight differences in food consumption of control and test item-treated females were considered to be incidental as they were not statistically significant and/or not dose-related. The overall differences in mean food consumption between control group and females treated with 100, 300 and 1000 mg/kg/day, respectively, were -4.7%, -2.0% and -3.4% during the pre-pairing phase, -3.4%, -3.0% and +4.4% during the gestation period, and -4.1%, -7.9% and +7.5% during the lactation period.

BODY WEIGHTS OF MALES

Body weights and body weight gain of males were considered to be unaffected by treatment with the test item.

Mean body weights of test item-treated males were similar to those in the control group during the whole treatment period. Mean body weight gain of males treated with 300 mg/kg/day was statistically significantly increased as compared to the control group during after-pairing. This was considered to be incidental as there was no dose-response relationship and the difference was only slight (+7% mean body weight gain during after pairing at 300 mg/kg/day vs. +4% in the control group).

BODY WEIGHTS OF FEMALES

No test item-related effects on body weights and body weight gain of females were observed.

Mean body weights and mean body weight gain of test item-treated females were similar to those of control females during the treatment period (pre-pairing to lactation).


2. REPRODUCTION AND BREEDING

MATING PERFORMANCE AND FERTILITY

No test item-related effect on mating performance or fertility was observed.

Except for one female at 300 mg/kg/day (no. 80), mating was recorded for all females during the first pairing period. Mean (median) precoital times calculated for the first pairing period were 2.1 (2), 2.7 (3), 3.1 (3) and 2.9 (3) days at 0, 100, 300 and 1000 mg/kg/day, respectively. The pre¬coital time of female no. 80 within the second pairing period was 2 days. The slight differences in precoital time between control and test item-treated females were considered to be incidental as there was no dose-response relationship.

Three females in the control group (nos. 52, 55 and 59), five treated with 300 mg/kg/day (nos. 73, 74, 75, 76 and 80), and two treated with the high dose 1000 mg/kg/day (88 and 92) were not pregnant. Fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 72.7%, 100%, 58.3% and 83.3% at 0, 100, 300 and 1000 mg/kg/day, respectively. The lower fertility index/conception rate in females treated with 300 mg/kg/day was considered to be incidental as there was no dose-response relationship.

The gestation index (number of females with living pups as a percentage of pregnant females) was 100% in females treated with 0, 100 or 300 mg/kg/day. At 1000 mg/kg/day the gestation index was 80% since two pregnant females (nos. 89 and 94) had died before delivery.

DURATION OF GESTATION

No test item-related effects on duration of gestation were observed. Mean duration of gestation was 21.8 days in the control group and 21.4 days in all test item-treated groups.

CORPORA LUTEA COUNT

No test item-related effects on corpora lutea count were observed. Mean number of corpora lutea per dam was 15.0, 14.6, 14.9 and 15.4 at 0, 100, 300 and 1000 mg/kg/day, respectively.

IMPLANTATION RATE AND POST IMPLANTATION LOSS

No test item-related effects on implantation rate and post-implantation loss were noted.

The mean number of implantations per dam was 12.3, 13.3, 13.7 and 14.5 at 0, 100, 300 and 1000 mg/kg/day, respectively.

The mean number of post-implantation loss per dam was 1.0, 0.8, 1.1 and 2.4 at 0, 100, 300 and 1000 mg/kg/day, respectively. Thus, the incidence of post-implantation loss was increased in the high dose group (1000 mg/kg/day) as compared to the control group: a total of 19 implantations in 4 litters (nos. 85, 86, 90 and 91) were lost compared to 8 implantations in 5 litters lost in the control group. The differences were not statistically significant. However, mean incidence of implantation loss per dam at the high dose level (2.4) was beyond the historical controls containing mean values from 0.7 to 1.4. Higher post-implantation loss was mainly due to two females (nos. 86 and 90); one of these females had 17 implantations and only 8 living pups. Therefore, the higher incidence of post-implantation loss was considered to be due to biological variability. Both the number of implantations and the litter size at 1000 mg/kg/day were higher than in the control group (see Section 4.3.6).

LITTER SIZE AT FIRST LITTER CHECK

No test item-related effects on litter size were noted.

The mean number of living pups per dam was 11.3, 12.4, 12.6 and 12.1 at 0, 100, 300 and 1000 mg/kg/day, respectively. The birth index (number of pups borne alive as a percentage of implantations) was 91.8%, 93.7%, 91.7% and 83.6% in these groups. Due to the slightly increased post-implantation loss during pregnancy, the birth index was lower at 1000 mg/kg/day as compared to the control group. It was also low as compared to the historical control data (range: 89.1% to 95.4%). However, the difference in birth index between control group and high dose group was not statistically significant and the number of living pups per dam was higher at 1000 mg/kg/day than in the control group. Therefore, the slightly lower birth index in the high dose group is considered to be incidental.

3. TERMINAL FINDINGS

ORGAN WEIGHTS

There were no test item-related effects on the weights of testes and epididymides in the parental males.


MACROSCOPICAL FINDINGS

- Males: There were no test item-related macroscopic findings in males.

All gross lesions observed in males at scheduled necropsy after 42 days of treatment with the test item were considered to be incidental as the findings or their microscopic correlate are within the range of normal background lesions. These findings were: pale discoloration of the liver in 1/12 (no. 18), 1/11 (no.35) and 2/12 (nos. 41 and 44) males treated with 100, 300 and 1000 mg/kg/day, respectively, reddish discoloration of the left seminal vesicle in one animal (no. 44) treated with 1000 mg/kg/day, and dark red foci on thymus and mandibular lymph node in one animal (no. 47) treated with 1000 mg/kg/day.

In the male that was killed for ethical reasons (no. 26 of Group 3, 300 mg/kg/day), cavity formation with watery fluid was observed at the brain.

- Females: There were no test item-related macroscopic findings in females.

No abnormal findings were recorded in any female that had not given birth (sacrifice on day 25 post coitum) and no gross lesions were observed in females of Group 4 (1000 mg/kg/day) that were sacrificed on day 4 post partum. Findings recorded in single females of Groups 2 or 3 (100 or 300 mg/kg/day) sacrificed on day 4 post partum were considered as incidental as they were isolated and there was no dose-response relationship. These findings were a light red watery cyst in the left ovary of female no. 72 (Group 2) and pelvic dilation in the left kidney of female no. 77, (Group 3).

The two females of Group 4 (1000 mg/kgday) that had died spontaneously on 24-Feb-2013 (during gestation) had both dark red discolored ovaries and embryos in the uterus. There was no indication for a dosing error.

Control female no. 51 that had died following an accident had dark red discolored lungs and several dark red foci on the thymus.

MICROSCOPIC FINDINGS

There were no test item-related microscopic findings.

Qualitative examination of the stages of spermatogenesis in the testis did not reveal any test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. No test item-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries.

The findings recorded were considered to be within the normal range of background alterations that is seen in untreated animals of this age and strain.

Dose descriptor:

NOEL

Remarks:

for general toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test-item related effects at 1000 mg/kg bw/day, the highest dose tested.

Dose descriptor:

NOEL

Remarks:

for reproduction

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test-item related effects at 1000 mg/kg bw/day, the highest dose tested

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:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

POSTNATAL LOSS DAYS 0 – 4 POST PARTUM

There was no test item-related effect on postnatal loss (days 0 - 4 post partum).

In the control group, 1 pup from one litter (no. 56) was found dead at first litter check. In addition, on day 2 post partum 2 pups from 2 litters (nos. 53 and 54) were missing in the control group. At 100 mg/kg/day, one pup each was missing on day 2 and day 4 post partum (litter nos. 68 and 64, respectively). At 300 mg/kg/day, 4 pups from litter no. 84 were missing on day 2 post partum. At the high dose of 1000 mg/kg/day, no postnatal loss was observed. The postnatal losses at 100 and 300 mg/kg/day were therefore considered as incidental.

The mean incidences of postnatal loss per dam were 0.3, 0.2, 0.6 and 0.0, and the mean litter size on day 4 post partum was 11.0, 12.3, 12.0 and 12.1 pups per dam at 0, 100, 300 and 1000 mg/kg/day, respectively.

EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK AND DURING LACTATION

No test item-related abnormalities were noted in pups during the first litter check or during lactation.

No milk in the stomach at first litter check in the only pup from litter no. 53 (missing on day 2 post partum) and a reddened tail apex in one pup from litter no. 56 were noted in the control group. Haematoma at the lower mandible in one pup from litter no. 62 at first litter check and a reddened tail apex in one pup from litter no. 64 were evident at 100 mg/kg/day. Both pups survived until scheduled necropsy. At 300 mg/kg/day, a missing tail apex was recorded for one pup (litter no. 84) on day 1 post partum, which was one out of four pups in this litter missing one day thereafter. No abnormality was noted in the high dose group.

PUP SEX RATIOS

Pups sex ratio was not affected by exposure to the test item.

At first litter check, the percentage of male pups was 56%, 44%, 51% and 43% at 0, 100, 300 and 1000 mg/kg/day, respectively. The slight differences in sex ratio between control group and groups 2 and 4 (100 and 1000 mg/kg/day, respectively), were considered to be incidental as there was no dose-response relationship and the data obtained for these dose groups are within the range of the historical control data.

BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUM

No test item-related effects on pup body weights and body weight gain were noted.

Mean body weights of pups (males and females together) on day 1 post partum were 6.0 g, 6.1 g, 6.0 g and 6.3 g, at 0, 100, 300 and 1000 mg/kg/day respectively. Mean body weight gain of pups during the first four days of the lactation period was +51.2%, +49.7%, +45.2% and +55.0%, respectively.

MACROSCOPIC FINDINGS OF PUPS

There were no abnormal findings at necropsy of pups.

Dose descriptor:

NOEL

Remarks:

for development

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test-item related effects at 1000 mg/kg bw/day, the highest dose tested

Reproductive effects observed:

not specified

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litter

 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	49 - 60	61 - 72	73 - 84	85 - 96
Number of females paired (A)	11	12	12	12
Number of females mated (B)	11	12	12	12
Number of paired females that died before the scheduled necropsy (C)	0	0	0	2
Number of females that were not pregnant (D)	3	0	5	2
Number of females giving birth to live pups (E)	8	12	7	8
Numbers of females that did not rear their pups until day 4post partum(F)	1	0	0	0
Number of females that reared their pups until day 4post partum	7	12	7	8

 

(A)  Female no. 51 died after an accident on day 12 of pre-pairing.

(B)  Female no. 80 was mated during the second mating period since no mating had been observed during the first one.

(C)  Female nos. 89 and 94 died prematurely on days 14 and 17, respectively, of the gestation period.

(D)  Female nos. 52, 55, 59 (control group), 73, 74, 75, 76 and 80 (300 mg/kg/day) as well as 88 and 92 (1000 mg/kg/day) were not pregnant. These animals were excluded from food consumption and body weight data summaries from the gestation period onwards.

(E)  Only females giving birth to live pups were included in the breeding data evaluation

(F)  Female no. 53 gave birth to one living pup but this pup was missing at the end of day 2 post partum.

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item DOBA to rats. DOBA was administered in corn oil as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. DOBA was administered to male rats for 42 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.

No test item-related effects were observed at any dose level including viability/mortality, food consumption, body weight development, clinical signs, organ weights, macroscopic and microscopic findings as well as reproduction and breeding data, i.e. mating performance and fertility, duration of gestation, corpora lutea count, implantation rate and post-implantation loss, litter size at first litter check and postnatal loss in the F0 generation. Pup data until day 4 post partum, i.e. external observations at first litter check and observations during lactation, sex ratios, pup body weight development and macroscopic findings were not affected by treatment with the test item.

Except for the mid dose group (300 mg/kg/day), there were at least 8 pregnant females per dose group that were used for evaluation of reproduction and development of offspring. Since no test item-related effects on these parameters were observed at the high dose level (1000 mg/kg/day), the low number of pregnancies in the mid dose group was considered to have no impact on the validity of this study.

Based on these results, the NOEL (No Observed Effect Level) for general toxicity in males and females as well as for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

GENERAL

 

The aim of this study was to assess the possible effect of DOBA on male, female fertility and embryofetal development in Wistar rat. This study was also aimed to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

 

Four groups of 12 males and 12 females were treated orally (by gavage) with DOBA once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy (in total 42 days). Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum or until day 24 post coitum if birth did not occur.

 

The following dose levels were used:

 

Group 1:     0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

 

After the treatment period (after 42 days for males, day 4 post partum for dams and pups or day 25 post coitum for females not giving birth), parent animals and pups were necropsied and examined macroscopically for any structural changes. Special attention was directed at the organs of the reproductive system. Testes and epididymides of all parental males were weighed separately. The number of implantation sites and corpora lutea was recorded for all dams with litters. Selected tissues were collected and fixed. The following tissues were examined by the study pathologist: ovaries, testes, epididymides, prostate and seminal vesicles from control animals, high dose animals and unscheduled deaths as well as all gross lesions.

 

 

MORTALITY OF PARENTAL ANIMALS

 

There was no test item-related effect on viability/mortality.

 

Three test item-treated animals died before scheduled termination: one male treated with 300 mg/kg/day was killed in extremis on day 5 after pairing because of a swelling in the neck region that was related to a lesion at the brain (cavity with watery fluid). In addition, two females treated with 1000 mg/kg/day died spontaneously during the gestation period. The death of these animals was considered related to the gavage procedure.  

 

CLINICAL SIGNS OF PARENTAL ANIMALS

 

No test item-related clinical signs were observed.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 

No test item-related effect on food consumption was noted in both genders.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 

Body weights and body weight gain of males and females were considered unaffected by treatment with the test item.

 

REPRODUCTION AND BREEDING DATA

 

No test item-related effects on mating performance, fertility, corpora lutea count, implantation rate, post-implantation loss, duration of gestation, litter size (at first litter check) or postnatal loss (days 0 - 4 post partum) were observed.

 

The incidence of post-implantation loss was increased and the birth index was decreased in the high dose group (1000 mg/kg/day) as compared to control group and historical control data. This was considered to be incidental since both the number of implantations and the litter size at 1000 mg/kg/day were higher than in the control group.

 

ORGAN WEIGHTS OF PARENTAL ANIMALS

 

There were no test item-related effects on the weights of testes and epididymides in the parental males.

 

MACROSCOPIC / MICROSCOPIC FINDINGS OF PARENTAL ANIMALS

 

There were no test item-related macroscopic or microscopic findings. Qualitative examination of the stages of spermatogenesis in the testis did not reveal any test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. No test item-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

No test item-related findings were noted in pups.

 

Pups sex ratio was not affected by the exposure to the test item.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 

No test item-related effects on pup body weights and body weight gain were noted.

 

MACROSCOPICAL FINDINGS IN PUPS

 

There were no abnormal findings at necropsy of pups.

 

 

CONCLUSION

 

Based on the results of this study, the NOEL (No Observed Effect Level) for general toxicity in males and females as well as for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restrictions - well performed GLP and OECD guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Short description of key information:
In a Reproduction/Developmental Toxicity Screening Test in the Han Wistar Rat the NOAEL for fertility was determined to be 1000 mg/kg bw., which was the higest dose tested. No effects were observed that could be associated with any toxicity to reproduction attributed to the test item.

Justification for selection of Effect on fertility via oral route:
most reliable and most specific study available

Effects on developmental toxicity

Description of key information

In a Reproduction/Developmental Toxicity Screening Test in the Han Wistar Rat the NOAEL for developmental toxicity was determined to be 1000 mg/kg bw., which was the higest dose tested. No effects were observed that could be associated with any developmental toxicity or teratogenicity attributed to the test item.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP and OECD Guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: Groups 1 to 4: 12 males and 12 females per group. Group 10 (reserve) 2 males and 2 females
- Total Number of Animals: 50 males and 50 females
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 249 to 390 g (males) and 205 to 229 g (females)
- Identification: Cage card and individual animal number (ear tattoo). On day 1 post partum, pups were individually tattooed with Indian ink.
- Randomization: Randomly allocated to groups by body weight.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temperature range: 20.5 - 23 °C; relative humidity range in general between 35% and 70% ). The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
- Accommodation: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 43/12) rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of respective samples were performed.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

DOSE FORMULATIONS

The dose formulations were not corrected for purity and were prepared as supplied by the Sponsor.

The dose formulations were prepared weekly as indicated by the results of stability analyses in the study D49720.

DOBA was weighed into a glass beaker on a tared Mettler balance. The vehicle was added and the mixtures were stirred using a magnetic stirrer and/or ultraturrax and used at room temperature (15 - 25 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STABILITY AND STORAGE OF DOSE FORMULATIONS

- Stability of Dose Formulations: For up to one week, based upon the results of stability analyses performed during Harlan Laboratories study D49720.
- Storage of Dose Formulations: At room temperature (20 ± 5 °C) in glass beakers.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
- Frequency of Administration: Daily
- Daily Target Dose Level: 0 mg/kg/day (Group 1), 100 mg/kg/day (Group 2), 300 mg/kg/day (Group 3) and 1000 mg/kg/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rats.
- Dose Volume: 5 mL/kg
- Dose Concentrations: 0 mg/mL/day (Group 1), 20 mg/mL/day (Group 2), 60 mg/mL/day (Group 3) and 200 mg/mL/day (Group 4)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD

The dose formulations were analyzed using a HPLC method provided by the Sponsor and adapted by Harlan Laboratories. The samples (approximately 1 g each) were delivered to the analytical laboratory.

Transport of Dose Formulations to Analytical Laboratory: At ambient temperature (20 ± 5 °C)
Storage of Dose Formulations in Analytical Laboratory: Frozen (-20 ± 5 °C)

Samples of dose formulations were not discarded without the written authorization of the study director. Solutions of samples were discarded latest 3 months after preparation without further notice.

The test item served as analytical standard/reference item.

Concentration and homogeneity of dose formulations were determined in samples taken as follows:

- Concentration and Homogeneity after Experimental Start (24-Jan-2013) and during Week 6 (28-Feb-2013): b (middle) in Group 1 and a (top), b (middle), c (bottom) in Groups 2 to 4.

RESULTS

The absence of the test item in the vehicle control samples (corn oil) was confirmed.

The DOBA concentrations in the dose formulations prepared for groups 2 - 4 ranged from 81.8% to 104.9% with reference to the nominal concentration. Thus, they were all within the accepted range of ±20% with reference to the nominal concentration.

The homogeneous distribution of DOBA in the preparations was approved because single results found did not deviate more than 3.7% from the corresponding mean and met the specified acceptance criterion of ≤15%.

In conclusion, the results indicate the accurate preparation of the test item DOBA in vehicle during this study.

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

As female no. 80 did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was carried out.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Duration of treatment / exposure:

42 days (males) and approximately 7 weeks (females)

Frequency of treatment:

Once daily

Duration of test:

MALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum per pairing period*
- Treatment Ends: On day before sacrifice
- Necropsy: After 42 days of treatment

FEMALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum per pairing period*
- Gestation: Approximately 21 days
- Treatment Ends: On day 3 post partum; females not giving birth on day 24 post coitum
- Necropsy: Dams and pups on day 4 post partum; females not giving birth on day 25 post coitum


* One female (no. 80) did not mate during the 14-day pairing period. Therefore, a second pairing of this female with a male in the same group, which had already mated successfully, was carried out. Since there was a second pairing period for one female, the length of the pairing phase was 15 days for all males.

Remarks:

Doses / Concentrations:
0, 100 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

The aim of this study was to assess the possible effect of DOBA on male, female fertility and embryofetal development in Wistar rat. This study was also aimed to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The test item was administered once daily orally (by gavage) to male and female rats throughout the pre-pairing and pairing periods, after pairing (males), gestation and lactation periods (females) including the day before scheduled necropsy.

This study should provide information to assess the need to conduct further investigations and may provide guidance in the design of subsequent studies.

Maternal examinations:

VIABILITY/MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations (once daily, during acclimatization and up to the day of necropsy). Additionally, females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

During pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum. No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

TERMINATION AND NECROPSY

Dams were sacrificed on day 4 post partum. When birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes. Special attention was directed at the organs of the reproductive system. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

TISSUE PRESERVATION

The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE

All preserved organ and tissue samples were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin.

HISTOPATHOLOGY

Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals which died spontaneously or had to be terminated in extremis.

Histological examination of ovaries was carried out on any females that did not give birth.

A histopathology phase report was provided by the principal investigator. A histopathology peer review was performed and the results included into the histopathology phase report.

Ovaries and uterine content:

The ovaries and the uterus were examined after termination on day 4 post partum. Examinations included the number of corpora lutea and the number of implantation sites.

Fetal examinations:

Not performed

Statistics:

The following statistical methods were used to analyze food consumption, body weights, organ weights, macroscopic findings, and reproduction data:

- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a 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 (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Indices:

From the on-line recorded reproduction data, the following parameters were calculated: mean precoital time, percentage mating, fertility index, conception rate, post-implantation loss, gestation index, birth index, dead/live pups at first litter check, pup sex ratio and viability index.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
1. IN-LIFE DATA

VIABILITY / MORTALITY

There was no test item-related effect on viability/mortality.

Female no. 51 of the control group died after an accident during application on day 12 of the pre-pairing period.

At 1000 mg/kg/day, female nos. 89 and 94 died prematurely on days 14 and 17, respectively, of the gestation period (both on 24-Feb-2013). Since both animals died on the same day and shortly after dosing, a correlation of these deaths to the gavage procedure seems probable, although no corresponding macroscopic findings were observed at necropsy. Therefore, the deaths of these animals are considered to be not related to the test item.

CLINICAL SIGNS OR OBSERVATIONS

No clinical signs were noted in females at any dose level.

FOOD CONSUMPTION OF FEMALES

No test item-related effect on food consumption was noted in females.

Slight differences in food consumption of control and test item-treated females were considered to be incidental as they were not statistically significant and/or not dose-related. The overall differences in mean food consumption between control group and females treated with 100, 300 and 1000 mg/kg/day, respectively, were -4.7%, -2.0% and -3.4% during the pre-pairing phase, -3.4%, -3.0% and +4.4% during the gestation period, and -4.1%, -7.9% and +7.5% during the lactation period.

BODY WEIGHTS OF FEMALES

No test item-related effects on body weights and body weight gain of females were observed.

Mean body weights and mean body weight gain of test item-treated females were similar to those of control females during the treatment period (pre-pairing to lactation).

2. TERMINAL FINDINGS

MACROSCOPICAL FINDINGS

There were no test item-related macroscopic findings in females.

No abnormal findings were recorded in any female that had not given birth (sacrifice on day 25 post coitum) and no gross lesions were observed in females of Group 4 (1000 mg/kg/day) that were sacrificed on day 4 post partum. Findings recorded in single females of Groups 2 or 3 (100 or 300 mg/kg/day) sacrificed on day 4 post partum were considered as incidental as they were isolated and there was no dose-response relationship. These findings were a light red watery cyst in the left ovary of female no. 72 (Group 2) and pelvic dilation in the left kidney of female no. 77, (Group 3).

The two females of Group 4 (1000 mg/kgday) that had died spontaneously on 24-Feb-2013 (during gestation) had both dark red discolored ovaries and embryos in the uterus. There was no indication for a dosing error.

Control female no. 51 that had died following an accident had dark red discolored lungs and several dark red foci on the thymus.

MICROSCOPIC FINDINGS

There were no test item-related microscopic findings.

No test item-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries.

The findings recorded were considered to be within the normal range of background alterations that is seen in untreated animals of this age and strain.

Dose descriptor:

NOEL

Remarks:

for general toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: effect type not specified

Dose descriptor:

NOEL

Remarks:

for development

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: effect type not specified

Dose descriptor:

NOEL

Remarks:

for reproduction

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: effect type not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Abnormalities:

not specified

Developmental effects observed:

not specified

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litter

 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	49 - 60	61 - 72	73 - 84	85 - 96
Number of females paired (A)	11	12	12	12
Number of females mated (B)	11	12	12	12
Number of paired females that died before the scheduled necropsy (C)	0	0	0	2
Number of females that were not pregnant (D)	3	0	5	2
Number of females giving birth to live pups (E)	8	12	7	8
Numbers of females that did not rear their pups until day 4post partum(F)	1	0	0	0
Number of females that reared their pups until day 4 post partum	7	12	7	8

 

(A)  Female no. 51 died after an accident on day 12 of pre-pairing.

(B)  Female no. 80 was mated during the second mating period since no mating had been observed during the first one.

(C)  Female nos. 89 and 94 died prematurely on days 14 and 17, respectively, of the gestation period.

(D)  Female nos. 52, 55, 59 (control group), 73, 74, 75, 76 and 80 (300 mg/kg/day) as well as 88 and 92 (1000 mg/kg/day) were not pregnant. These animals were excluded from food consumption and body weight data summaries from the gestation period onwards.

(E)  Only females giving birth to live pups were included in the breeding data evaluation.

(F)  Female no. 53 gave birth to one living pup but this pup was missing at the end of day 2 post partum.

 

MATING PERFORMANCE AND FERTILITY

 

No test item-related effect on mating performance or fertility was observed.

 

Except for one female at 300 mg/kg/day (no. 80), mating was recorded for all females during the first pairing period. Mean (median) precoital times calculated for the first pairing period were 2.1 (2), 2.7 (3), 3.1 (3) and 2.9 (3) days at 0, 100, 300 and 1000 mg/kg/day, respectively. The pre¬coital time of female no. 80 within the second pairing period was 2 days. The slight differences in precoital time between control and test item-treated females were considered to be incidental as there was no dose-response relationship.

 

Three females in the control group (nos. 52, 55 and 59), five treated with 300 mg/kg/day (nos. 73, 74, 75, 76 and 80), and two treated with the high dose 1000 mg/kg/day (88 and 92) were not pregnant. Fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 72.7%, 100%, 58.3% and 83.3% at 0, 100, 300 and 1000 mg/kg/day, respectively. The lower fertility index/conception rate in females treated with 300 mg/kg/day was considered to be incidental as there was no dose-response relationship.

 

The gestation index (number of females with living pups as a percentage of pregnant females) was 100% in females treated with 0, 100 or 300 mg/kg/day. At 1000 mg/kg/day the gestation index was 80% since two pregnant females (nos. 89 and 94) had died before delivery. 

 

DURATION OF GESTATION

 

No test item-related effects on duration of gestation were observed. Mean duration of gestation was 21.8 days in the control group and 21.4 days in all test item-treated groups.

 

CORPORA LUTEA COUNT

 

No test item-related effects on corpora lutea count were observed. Mean number of corpora lutea per dam was 15.0, 14.6, 14.9 and 15.4 at 0, 100, 300 and 1000 mg/kg/day, respectively.

 

IMPLANTATION RATE AND POST IMPLANTATION LOSS

 

No test item-related effects on implantation rate and post-implantation loss were noted.

 

The mean number of implantations per dam was 12.3, 13.3, 13.7 and 14.5 at 0, 100, 300 and 1000 mg/kg/day, respectively.

 

The mean number of post-implantation loss per dam was 1.0, 0.8, 1.1 and 2.4 at 0, 100, 300 and 1000 mg/kg/day, respectively. Thus, the incidence of post-implantation loss was increased in the high dose group (1000 mg/kg/day) as compared to the control group: a total of 19 implantations in 4 litters (nos. 85, 86, 90 and 91) were lost compared to 8 implantations in 5 litters lost in the control group. The differences were not statistically significant. However, mean incidence of implantation loss per dam at the high dose level (2.4) was beyond the historical controls containing mean values from 0.7 to 1.4. Higher post-implantation loss was mainly due to two females (nos. 86 and 90); one of these females had 17 implantations and only 8 living pups. Therefore, the higher incidence of post-implantation loss was considered to be due to biological variability. Both the number of implantations and the litter size at 1000 mg/kg/day were higher than in the control group (see Section 4.3.6).

 

LITTER SIZE AT FIRST LITTER CHECK

 

No test item-related effects on litter size were noted.

 

The mean number of living pups per dam was 11.3, 12.4, 12.6 and 12.1 at 0, 100, 300 and 1000 mg/kg/day, respectively. The birth index (number of pups borne alive as a percentage of implantations) was 91.8%, 93.7%, 91.7% and 83.6% in these groups. Due to the slightly increased post-implantation loss during pregnancy, the birth index was lower at 1000 mg/kg/day as compared to the control group. It was also low as compared to the historical control data (range: 89.1% to 95.4%). However, the difference in birth index between control group and high dose group was not statistically significant and the number of living pups per dam was higher at 1000 mg/kg/day than in the control group. Therefore, the slightly lower birth index in the high dose group is considered to be incidental. 

 

POSTNATAL LOSS DAYS 0 – 4 POST PARTUM

 

There was no test item-related effect on postnatal loss (days 0 - 4 post partum).

 

In the control group, 1 pup from one litter (no. 56) was found dead at first litter check. In addition, on day 2 post partum 2 pups from 2 litters (nos. 53 and 54) were missing in the control group. At 100 mg/kg/day, one pup each was missing on day 2 and day 4 post partum (litter nos. 68 and 64, respectively). At 300 mg/kg/day, 4 pups from litter no. 84 were missing on day 2 post partum. At the high dose of 1000 mg/kg/day, no postnatal loss was observed. The postnatal losses at 100 and 300 mg/kg/day were therefore considered as incidental.

 

The mean incidences of postnatal loss per dam were 0.3, 0.2, 0.6 and 0.0, and the mean litter size on day 4 post partum was 11.0, 12.3, 12.0 and 12.1 pups per dam at 0, 100, 300 and 1000 mg/kg/day, respectively.

 

EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

No test item-related abnormalities were noted in pups during the first litter check or during lactation.

 

No milk in the stomach at first litter check in the only pup from litter no. 53 (missing on day 2 post partum) and a reddened tail apex in one pup from litter no. 56 were noted in the control group. Haematoma at the lower mandible in one pup from litter no. 62 at first litter check and a reddened tail apex in one pup from litter no. 64 were evident at 100 mg/kg/day. Both pups survived until scheduled necropsy. At 300 mg/kg/day, a missing tail apex was recorded for one pup (litter no. 84) on day 1 post partum, which was one out of four pups in this litter missing one day thereafter. No abnormality was noted in the high dose group.

 

PUP SEX RATIOS

 

Pups sex ratio was not affected by exposure to the test item.

 

At first litter check, the percentage of male pups was 56%, 44%, 51% and 43% at 0, 100, 300 and 1000 mg/kg/day, respectively. The slight differences in sex ratio between control group and groups 2 and 4 (100 and 1000 mg/kg/day, respectively), were considered to be incidental as there was no dose-response relationship and the data obtained for these dose groups are within the range of the historical control data.

 

BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUM

 

No test item-related effects on pup body weights and body weight gain were noted.

 

Mean body weights of pups (males and females together) on day 1 post partum were 6.0 g, 6.1 g, 6.0 g and 6.3 g, at 0, 100, 300 and 1000 mg/kg/day respectively. Mean body weight gain of pups during the first four days of the lactation period was +51.2%, +49.7%, +45.2% and +55.0%, respectively.

 

MACROSCOPIC FINDINGS OF PUPS

 

There were no abnormal findings at necropsy of pups.

 

2. IN-LIFE DATA OF PARENTAL MALES

 

VIABILITY / MORTALITY

 

There was no test item-related effect on viability/mortality.

 

Male no. 26 (300 mg/kg/day) was killed in extremis on day 5 after pairing because of swelling in the neck region that made a correct test item-administration impossible. At necropsy, a lesion at the brain (cavity with watery fluid) was noted. 

 

CLINICAL SIGNS OR OBSERVATIONS

 

No test item-related clinical signs were observed.

 

A kinked tail apex was recorded for male no. 27 (300 mg/kg/day) from day 10 of the pre-pairing period onwards. In male no. 26 (300 mg/kg/day), swelling in the neck region and slightly tilted head was noted during the after pairing phase. The animal was subsequently killed for ethical reasons.

 

No further clinical signs were noted in males at any dose level.

 

FOOD CONSUMPTION OF MALES

 

No test item-related effect on food consumption was noted in males.

 

The overall differences in mean food consumption between control group and males treated with 100, 300 and 1000 mg/kg/day, respectively, were 1.8%, -5.0% and +0.9% during the pre-paring phase, and +4.5%, +2.5% and +1.0%, during the after-pairing phase.

 

BODY WEIGHTS OF MALES

 

Body weights and body weight gain of males were considered to be unaffected by treatment with the test item.

 

Mean body weights of test item-treated males were similar to those in the control group during the whole treatment period. Mean body weight gain of males treated with 300 mg/kg/day was statistically significantly increased as compared to the control group during after-pairing. This was considered to be incidental as there was no dose-response relationship and the difference was only slight (+7% mean body weight gain during after pairing at 300 mg/kg/day vs. +4% in the control group).

 

3. TERMINAL FINDINGS IN PARENTAL MALES

 

ORGAN WEIGHTS

 

There were no test item-related effects on the weights of testes and epididymides in the parental males.

 

MACROSCOPICAL FINDINGS

 

There were no test item-related macroscopic findings in males.

 

All gross lesions observed in males at scheduled necropsy after 42 days of treatment with the test item were considered to be incidental as the findings or their microscopic correlate are within the range of normal background lesions. These findings were: pale discoloration of the liver in 1/12 (no. 18), 1/11 (no.35) and 2/12 (nos. 41 and 44) males treated with 100, 300 and 1000 mg/kg/day, respectively, reddish discoloration of the left seminal vesicle in one animal (no. 44) treated with 1000 mg/kg/day, and dark red foci on thymus and mandibular lymph node in one animal (no. 47) treated with 1000 mg/kg/day.    

 

In the male that was killed for ethical reasons (no. 26 of Group 3, 300 mg/kg/day), cavity formation with watery fluid was observed at the brain.

 

MICROSCOPIC FINDINGS

 

There were no test item-related microscopic findings.

 

Qualitative examination of the stages of spermatogenesis in the testis did not reveal any test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle.

 

The findings recorded were considered to be within the normal range of background alterations that is seen in untreated animals of this age and strain.

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item DOBA to rats. DOBA was administered in corn oil as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. DOBA was administered to male rats for 42 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.

No test item-related effects were observed at any dose level including viability/mortality, food consumption, body weight development, clinical signs, organ weights, macroscopic and microscopic findings as well as reproduction and breeding data, i.e. mating performance and fertility, duration of gestation, corpora lutea count, implantation rate and post-implantation loss, litter size at first litter check and postnatal loss in the F0 generation. Pup data until day 4 post partum, i.e. external observations at first litter check and observations during lactation, sex ratios, pup body weight development and macroscopic findings were not affected by treatment with the test item.

Except for the mid dose group (300 mg/kg/day), there were at least 8 pregnant females per dose group that were used for evaluation of reproduction and development of offspring. Since no test item-related effects on these parameters were observed at the high dose level (1000 mg/kg/day), the low number of pregnancies in the mid dose group was considered to have no impact on the validity of this study.

Based on these results, the NOEL (No Observed Effect Level) for general toxicity in males and females as well as for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

GENERAL

 

The aim of this study was to assess the possible effect of DOBA on male, female fertility and embryofetal development in Wistar rat. This study was also aimed to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

 

Four groups of 12 males and 12 females were treated orally (by gavage) with DOBA once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy (in total 42 days). Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum or until day 24 post coitum if birth did not occur.

 

The following dose levels were used:

 

Group 1:     0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

 

After the treatment period (after 42 days for males, day 4 post partum for dams and pups or day 25 post coitum for females not giving birth), parent animals and pups were necropsied and examined macroscopically for any structural changes. Special attention was directed at the organs of the reproductive system. Testes and epididymides of all parental males were weighed separately. The number of implantation sites and corpora lutea was recorded for all dams with litters. Selected tissues were collected and fixed. The following tissues were examined by the study pathologist: ovaries, testes, epididymides, prostate and seminal vesicles from control animals, high dose animals and unscheduled deaths as well as all gross lesions.

 

 

MORTALITY OF PARENTAL ANIMALS

 

There was no test item-related effect on viability/mortality.

 

Three test item-treated animals died before scheduled termination: one male treated with 300 mg/kg/day was killed in extremis on day 5 after pairing because of a swelling in the neck region that was related to a lesion at the brain (cavity with watery fluid). In addition, two females treated with 1000 mg/kg/day died spontaneously during the gestation period. The death of these animals was considered related to the gavage procedure.  

 

CLINICAL SIGNS OF PARENTAL ANIMALS

 

No test item-related clinical signs were observed.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 

No test item-related effect on food consumption was noted in both genders.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 

Body weights and body weight gain of males and females were considered unaffected by treatment with the test item.

 

REPRODUCTION AND BREEDING DATA

 

No test item-related effects on mating performance, fertility, corpora lutea count, implantation rate, post-implantation loss, duration of gestation, litter size (at first litter check) or postnatal loss (days 0 - 4 post partum) were observed.

 

The incidence of post-implantation loss was increased and the birth index was decreased in the high dose group (1000 mg/kg/day) as compared to control group and historical control data. This was considered to be incidental since both the number of implantations and the litter size at 1000 mg/kg/day were higher than in the control group.

 

ORGAN WEIGHTS OF PARENTAL ANIMALS

 

There were no test item-related effects on the weights of testes and epididymides in the parental males.

 

MACROSCOPIC / MICROSCOPIC FINDINGS OF PARENTAL ANIMALS

 

There were no test item-related macroscopic or microscopic findings. Qualitative examination of the stages of spermatogenesis in the testis did not reveal any test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. No test item-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

No test item-related findings were noted in pups.

 

Pups sex ratio was not affected by the exposure to the test item.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 

No test item-related effects on pup body weights and body weight gain were noted.

 

MACROSCOPICAL FINDINGS IN PUPS

 

There were no abnormal findings at necropsy of pups.

 

 

CONCLUSION

 

Based on the results of this study, the NOEL (No Observed Effect Level) for general toxicity in males and females as well as for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restrictions - well performed GLP and OECD guideline study

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

Justification for selection of Effect on developmental toxicity: via oral route:
most reliable and most specific study available

Justification for classification or non-classification

In accordance with Section 1.2 of REACH Annex XI, there is sufficient weight of evidence from several independent sources of information leading to the conclusion that the test item (DOBA) does neither cause developmental toxicity nor toxicity to reproduction and thus doesn't have to be classified, because

- the substance did not cause lethal effects or signs of systemic toxicity after administration of a single oral or dermal dose of 2000 mg/kg in tests for acute toxicity in rats,

- the substance is not irritating or corrosive and has not to be classified as eye or skin irritant, based on findings from in vivo tests for skin and eye irritation/corrosion,

- the substance does not have to be classified as skin sensitizing based on the findings in a Local Lymh Node Assays in mice, and a Guinea Pig Maximistaion Test,

- the substance caused no relevant systemic toxic effects in a subacute oral toxicity study in rats comprising in addition to standard examinations sperm analysis and vaginal estrus stages (NOAEL: 1000 mg/kg/day, no adverse effects up to the highest dose tested)

- the substance caused no systemic toxic effects, and no effects on fertility or development in a Reproduction / Developmental Toxicity Screening Test (OECD Guideline 421; NOAEL: 1000 mg/kg bw/day, no adverse effects up to the highest dose tested),

- a QSAR analysis (DEREK NEXUS^®) disclosed no structural segments that are suspected to cause developmental toxicity or toxicity to reproduction in any mammalian species,

- the absence of effects in the above-mentioned toxicity endpoint tests indicates that the substance does not adversly interact with living cells or tissues after oral and dermal exposure for up to 42 days,

It can therefore be concluded with sufficient certainty and without further testingt that the substance will not cause developmental toxicity or toxicity to reproduction.