Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

For the assessment of the reproduction toxicity of the registration substance two key studies are to be used: one reproduction toxicity screening study (OECD 421) on the registration substance and one 2 -generation study (OECD 416) on the read-across source substance.

 

The reproduction toxicity of the registration substance was investigated according to the OECD 421.

The rats were treated with the registration substance daily per gavage at doses of 0, 10, 30 and 75 mg/kg/d. Males were treated two weeks prior to mating, throughout mating and until one day before necropsy, total treatment period being at least 29 days. Females were treated two weeks prior to mating, throughout mating, gestation and parturition and up to the post partum day 4.

Reduced body weight gain was found for the animals treated at 75 mg/kg/d. No significant treatment related effect was found for the reproduction performance up to the dose of 75 mg/kg/d. The pups at 75 mg/kg/d exhibited reduced viability and reduced body weight.

NOAEL 30 mg/kg/d for the systemic toxicity of parental animals, NOAEL 75 mg/kg/d for the reproduction performance and NOAEL 30 mg/kg/d for the developmental toxicity was obtained.

 

In a two-generation study, C12-16 ADBAC was administered in the diet to male and female Sprague Dawley rats at 0, 500, 2,000 or 4,000 ppm before and through mating and gestation until the end of the lactation period in both F0 and F1 generations.

The actual F0 intake of C12 -C16 ADCBA throughout the study was 8, 30.5 and 61.5 mg/kg/d for males and 9 -18.5, 34.5 -79.5 and 72.5 -163 mg/kg/d for females. And the actual F1 intake of C12 -C16 ADCBA throughout the study was 12, 48 and 101 mg/kg/d for males and 10.5 -20.5, 41.5 -81 and 82 -161.5 mg/kg/d for females.

At 2,000 ppm, F0 (males) and F1 parents showed marginally to slightly lower body weight gains and reduced food consumption. Necropsy of parents of both generations revealed dilatation of the caecum in some animals.

At 4,000 ppm, in F0 and F1 parents, number of implantation sites and litter size at birth were reduced. The progeny (F2) also showed lower pup weights. Pup weight gain was slightly lower during lactation. Upon necropsy, dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females.

Treatment with the test substance had no effect on mating, fertility and behavioural parameters in F0 and F1 parental Sprague-Dawley rats at treatment levels up to 2,000 ppm. No effect was recorded on litter parameters and on pre- and post-natal development of either generation at 2,000 ppm.

The NOAEL for parental toxicity was 2000 ppm (equivalent to ca. 50 mg/kg/d),  the NOAEL for developmental toxicity was 4000 ppm (equivalent to ca- 50 mg/kg/d) and the NOAEL for fertility was 4000 ppm (equivalent to ca. 100 mg/kg/d).

 

In both studies comparable results were obtained: reduced body weight gains were found in both studie as primary systemic effect and reproduction/developmental effects were found only in presense of systemic effects. Also the NOAELs obtained in both studies are in the same dose range, indicating comparable potency with respect to systemic and reproduction/developmental toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2009-10-20 to 2010-05-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
Justification for the animal treatment per gavage and for the top dose of 75 mg/kg/d for the main study:
Prior to this main study a dose range finding was performed. The rats were treated either via gavage or via feeding, which was to find out whether the feeding would reduce the irritation effect that was found in the 28-day toxicity study. No significant reduction of toxicity was found when the animals were treated via feeding.
The animals treated at 150 mg/kg/d via gavage exhibited severe toxicity, which lead to the determination of 75 mg/kg/d as top dose for the main study.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Animals: Rat, HanRcc: 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: 40 males (10 per group) and 40 females (10 per group)
Age (at Start of Treatment): 11 weeks
Body Weight Range (at Start of Treatment): Males (286 to 330 g) and females (181 to 205 g)
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation were taken into consideration in order to ensure similar mean body weights in all groups.
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 (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: 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). 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 Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 60/09).
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

C20/22 ATQ trocken was weighed into a plastic dish on a tared precision balance, transferred in a brown glass and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.


STORAGE OF DOSE FORMULATIONS

Dose formulations were stored at room temperature (20 ± 5 °C) in brown glass beakers.

Based upon the results of stability analyses performed within the Harlan Laboratories study C40763, non-GLP, dose formulations were stable for at least one week.


TREATMENT

Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
Frequency of Administration: Once daily
Target Dose Levels: Group 1: 0 mg/kg/day (control group); Group 2: 10 mg/kg/day; Group 3: 30 mg/kg/day; Group 4: 75 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study C40763, where the administration of the test item was by gavage and by diet. The dose levels used where 50 and 150 mg/kg/ day as well as 600, 1800 and 6000 ppm. The groups receiving 150 mg/kg/day, 1800 or 6000 ppm had to be sacrificed for ethical reasons before the planned study termination due to marked body weight loss and general bad conditions. The NOAEL was considered to be 50 mg/kg/day or 600 ppm.
Dose Volume: 10 mL/kg body weight
Duration of Acclimatization Period: 9 days
Duration of Treatment Period: Males (minimum 4 weeks); females (approximately 7 weeks)
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 of mating was designated day 0 post coitum.

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:
ANALYSIS OF DOSE FORMULATIONS

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 hrs and 6 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Analytical Department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an ELSD detector following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

The application formulations investigated during the study were found to comprise C20/22-ATQ trocken in the range of 94.5% to 106.9% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of C20/22-ATQ trocken in the preparations was approved because single results found did not deviate more than 4.2% (<15%) from the corresponding mean. In addition, the test item was found to be stable in application formulations when kept 6 days at room temperature (20±5°C) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.
Duration of treatment / exposure:
Males: Minimum 4 weeks
Females: Approximately 7 weeks
Frequency of treatment:
Once daily
Details on study schedule:
Acclimatization: 9 days (males and females)
First Test Item Administration : Day 1 of pre-pairing (males and females)
Pre-Pairing: 14 days (males and females)
Pairing: 14 days maximum (males and females)
Gestation: Approximately 21 days (females)
Treatment Ends: On day before sacrifice (males); on day 3 post partum (females)
Necropsy: After a minimum of 28 days treatment (males); on day 4 post partum (females)
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to 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 period); females (pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and 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.
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 OF THE STUDY

Males were sacrificed after they had been treated for at least 28 days. Dams were sacrificed on day 4 post partum.

If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.


NECROPSY

At the scheduled sacrifice, all parent animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes at the scheduled necropsy.

For the parent animals, special attention was directed at the organs of the reproductive system.
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 as pairs.


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 organ and tissue samples to be examined by the study pathologist 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 study pathologist.

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. In addition, microscopic examination of the reproductive organs of all infertile males was made.

A peer review was carried out by the Department of Pathology, Harlan Laboratories Ltd, Itingen / Switzerland.

Postmortem examinations (offspring):
TERMINATION OF THE STUDY

Pups were sacrificed on day 4 post partum.


NECROPSY

At the scheduled sacrifice, all pups were killed by an injection of sodium pentobarbital.

Dead pups, except those excessively cannibalized, were examined macroscopically.

All pups were examined macroscopically for any structural changes at the scheduled necropsy.

Statistics:
The following statistical methods were used to analyze food consumption, body weights, reproduction data, organ weights and macroscopic findings:
- 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: fertility indices, mean precoital time, post-implantation losses, mean litter size.
Offspring viability indices:
From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex ratios and postnatal loss (up to day 4 post partum).
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males at 75 and 30 mg/kg/day and in females at 75 mg/kg bw/day food consumption was decreased. In males and females at the dose level of 75 mg/kg bw/day body weights and body weight gain were decreased.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males at 75 and 30 mg/kg/day and in females at 75 mg/kg bw/day food consumption was decreased. In males and females at the dose level of 75 mg/kg bw/day body weights and body weight gain were decreased.
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:
no effects observed
Description (incidence and severity):
by means of testis and epididymis weights, histopathological examination of of testes, epididymides, prostate and seminal vesicles with special emphasis on the stages of spermatogenesis and histopathology of interstitial cell structure.
Reproductive performance:
no effects observed
1 IN-LIVE DATA - PARENTAL ANIMALS

1.1 CLINICAL SIGNS OR OBSERVATIONS
All animals survived until the scheduled necropsy.
In group 4, ruffled fur was noted in female no. 72 on day 22 of the gestation until day 2 of lactation, in female no. 73 on day 17 of gestation and in female no. 75 on days 2, 3 and 4 of lactation period. Vaginal bleeding was observed in female no. 79 on days 16 and 17 of gestation. These clinical signs were considered to be incidental since they were occasionally noted in single females.

1.2 FOOD CONSUMPTION OF MALES
Pre-pairing Period:
In groups 3 and 4, mean food consumption was dose-dependently and statistically significantly decreased during the entire pre-pairing period (-11.0% and -19.4% compared to the control, respectively). This was considered to be due to the treatment with the test item.
In group 2, no test item-related effects were noted.

1.3 FOOD CONSUMPTION OF FEMALES
Pre-pairing, Gestation and Lactation Periods:
In group 4, during the pre-pairing period mean food consumption was lower during the first week (-12.0% compared to the control). During the gestation period, mean food consumption started to decrease on days 7 - 14 (-7.4% compared to the control) followed by a marked decrease on days 14 - 21 (-17.8% compared to the control) through the lactation period (-20.2% compared to the control). These reductions were considered to be test item-related although they only attained statistical significance on the first week of the pre-pairing and during the last week of gestation period.
In groups 2 and 3, no test item-related effects were noted. The lower differences which occurred occasionally in group 3 were considered to be incidental.

1.4 BODY WEIGHTS OF MALES
Pre-pairing and Pairing Periods:
In group 4, during the pre-pairing period mean body weight was statistically significantly lower starting on day 7 onwards and mean body weight gain was statistically significantly lower during the entire pre-pairing period (weight gain: +3.3% versus +10.1%). This was considered to be a test item-related effect. During the pairing period, mean body weight remained statistically significantly lower while mean body weight gain was similar to the control (weight gain: +8.8% versus +10.5%).
In groups 2 and 3, no test item-related effects were observed in body weight and body weight gain.

1.5 BODY WEIGHTS OF FEMALES
Pre-pairing, Gestation and Lactation Periods:
In group 4, during the pre-pairing period mean body weight gain was statistically significantly decreased starting on day 9 until the end of the period while mean body weight was not affected (overall weight gain 4.7% versus +6.8%). During the gestation period, mean body weight was statistically significantly lower between days 16 and 21 and mean body weight gain was statistically significantly lower at the end of the period (overall weight gain +44.9% versus +56.6%). During the lactation period, mean body weight was statistically significantly lower for the whole period while mean body weight gain was not affected.
In groups 2 and 3, no test item-related effects were noted.

1.6 MATING PERFORMANCE AND FERTILITY
The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 1.9, 3.0, 2.6 and 2.1 days in order of ascending dose level. The median precoital time was 2, 3, 3 and 2 days in order of ascending dose level. In group 4, one female mated on the first day on the second pairing period.
One female (no. 42) in group 1 was not pregnant. Thus the fertility indices were 90.0% in group 1 and 100.0% in groups 2, 3 and 4.

1.7 DURATION OF GESTATION
The mean duration of gestation was unaffected by treatment with the test item. Mean duration of gestation was 21.4, 21.4, 21.6 and 21.6 days, in order of ascending dose level.

1.8 CORPORA LUTEA COUNT
The mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (13.9, 13.7, 13.3 and 13.3 in order of ascending dose level) and gave no indication of any test item-related effect.

1.9 IMPLANTATION RATE AND POST-IMPLANTATION LOSS
The mean number of implantations per dam was not affected by the treatment with the test item. The mean numbers of implantations per litter were 13.4, 13.5, 11.4 and 12.2 in order of ascending dose level. In group 3, the lower mean number of implantations was considered to be incidental since it did not follow a dose-dependent pattern and the value in group 4 was within the range of the historical control data.
Mean incidence of post-implantation loss as a percentage of total implantations was 8.3, 8.1, 12.3 and 17.3% in order of ascending dose level. In group 4, total number of post-implantation losses was statististically significantly higher. This was due to one dam (no. 72) which had total post-implantation loss (three post-implantation losses and ten pups found dead at first litter check). Since this occurred in one single female, post-implantation loss was not considered to be affected by the treatment with the test item.

1.10 LITTER SIZE AT FIRST LITTER CHECK
The number of live pups at first litter check was unaffected by treatment with the test item. The mean number of live pups per litter was 12.3, 12.4, 10.0 and 10.1 in order of ascending dose level and within the range of the historical control data. One dam (no. 72) in group 4 cannibalized all its ten pups before first litter check.
Birth index was 91.7, 91.9, 87.7 and 82.7% in order of ascending dose level. The statistically significantly lower birth index in group 4, which was due to whole litter loss of one single animal (no. 72) was still within the range of the historical control data.

1.11 POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
In group 4, total postnatal loss was statistically significantly higher (seven pups versus 0 in the control). This was due to one dam (no. 75), which did not consume any food during the lactation period and which lost five pups on day 4 post partum. The postnatal loss was within the range of the historical control data and therefore not considered to be test item-related.
For females, which reared their pups until day 4 post partum, mean litter size on day 4 post partum was 12.3, 12.4, 10.0 and 10.5 pups in groups 1, 2, 3 and 4, respectively. Lower litter size in group 3 was secondary to the lower number of implantation sites.
The resulting viability indices were 100.0%, 100.0%, 100.0% and 92.3% in order of ascending dose levels. The statistically significantly lower viability index in group 4 was within the range of the historical control data.


2 TERMINAL FINDINGS - PARENTAL ANIMALS

2.1 ORGAN WEIGHTS
Absolue and relative weight of testes and epididymides were not affected by the treatment with the test item.

2.2 MACROSCOPICAL FINDINGS
In group 4, size of both testes and epdidimides were reduced in one male. One female was noted to have both uterine horns discolored dark-red and with firm reddish nodules in the mucosa of the right horn. In group 3, one female had left uterine horn dilated with mucous. Since these findings occurred isolated in single animals, they were not considered to be test item-related.

2.3 HISTOPATHOLOGY FINDINGS
Marked tubular atrophy of the testes and massive hypospermia of the epididymides were recorded in one male (no. 39) of group 4. These findings were considered to be spontaneous changes and not to be test item-related lesions, because minimal tubular atrophy of the testes was also recorded in one male of the control group and there were neither further findings of testes toxicity nor sperm staging abnormality in other males of group 4. All other findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. No test item-related histological finding was recorded in ovary from two females (no. 42, 79) that did not give birth.

Sperm staging:
No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
30 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
reproductive performance
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 75 mg/kg/day, pup weight gain and pup weight on day 4 post partum were reduced.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
1 IN-LIVE DATA - F1 PUPS

1.1 EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION
In group 4, all pups in litter no. 72 were found dead. All these pups were cannibalized and/or in advanced autolysis. No other external findings were noted, therefore these findings were considered to be incidental.

1.2 SEX RATIOS
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item.
The proportion of males on day 4 post partum was 54%, 51%, 42% and 44% in order of ascending dose level.

1.3 PUP WEIGHTS TO DAY 4 POST PARTUM
The mean pup weights were were similar to the control group. On day 1 post partum mean pup weights were 5.7, 5.9, 5.9 and 5.5 g for combined data of male and female pups in order of ascending dose level.
In group 4, mean pup weight gain up to day 4 postpartum was lower than the control group (+29.1% versus +38.6%) and the mean pup weight was lower on day 4 postpartum (7.1 g versus 7.9 g). These differences were not statistically significant and most likely due to nursing problems based on lower food consumption of the group 4 females during lactation. In groups 2 and 3, mean pup weight gain up to day 4 post partum were unaffected by the treatment with the test item. Mean pup weights on day 4 postpartum were 8.3 and 8.4 g compared to 7.9 g in the control group for combined data of male and female pups.

1.4 MACROSCOPICAL FINDINGS
No relevant abnormal findings were noted at macroscopic examination of the pups. In group 4, all pups in one litter which were found dead at first litter check were cannibalized and/or advanced autolysed. No other findings were noted.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Key result
Reproductive effects observed:
no

   

Table 1: Animals Breeding for F1 Litters

 

Control

10 mg/kg/d

30 mg/kg/d

75 mg/kd/d

Female numbers

41-50

51-60

61-70

71-80

Number of females paired

10

10

10

10

Number of females mated

10

10

10

10

Number of pregnant females (A)

9

10

10

10

Number of females with only implantation sites (B)

 

 

 

1

Numbers of females which cannibalized the whole litter (C)

 

 

 

1

Number of females which reared

their pups until day 4 post partum

9

10

10

8

A) Female No. 42 was not pregnant

B) Female No. 79 had only implantation sites

C) Female No. 72 had cannibalized the whole litter at first litter check

 

Table 2a: Body weight of males [g] (n = 10)

 

Control

10 mg/kg/d

30 mg/kg/d

75 mg/kg/d

Pre-mating

 

 

 

 

Day 1

308±7.5

304± 13.7

300± 6.0

305± 13.0

Day 8

325± 8.2

323± 15.5

312± 11.8

*306± 17.2

Day 14

339± 8.5

342± 16.0

325± 15.8

**315± 23.6

Mating

 

 

 

 

Day 1

334± 10.3

338± 16.3

320± 16.6

**306± 27.4

Day 8

351± 12.2

354± 15.6

332± 16.2

**319± 32.5

Day 15

369± 13.5

375± 18.0

350± 19.6

**333± 31.1

 

 

 

Table 2b: Body weight of females [g]

 

Control

10 mg/kg/d

30 mg/kg/d

75 mg/kg/d

Pre-mating

 

 

 

 

Day 1

192± 5.4

(n = 10)

193± 6.9

(n = 10)

189± 5.6

(n = 10)

192± 4.2

(n = 10)

Day 8

197± 3.0

(n = 10)

204± 7.6

(n = 10)

195± 8.0

(n = 10)

194± 8.7

(n = 10)

Day 14

205± 5.2

(n = 10)

210± 8.8

(n = 10)

200± 8.6

(n = 10)

201± 7.8

(n = 10)

Gestation

 

 

 

 

Day 1

205± 4.7

(n = 9)

211± 8.9

(n = 10)

198± 6.8

(n = 10)

198± 7.3

(n = 9)

Day 8

230± 7.2

(n = 9)

239± 10.4

(n = 10)

222± 11.6

(n = 10)

226± 9.2

(n = 9)

Day 15

260± 8.3

(n = 9)

268± 11.3

(n = 10)

248± 11.7

(n = 10)

247± 14.7

(n = 9)

Day 21

321± 13.4

(n = 9)

331± 13.8

(n = 10)

*299± 17.4

(n = 10)

**287± 28.8

(n = 9)

Lactation

 

 

 

 

Day 1

230± 9.9

(n = 9)

239± 12.2

(n = 10)

221± 13.9

(n = 10)

*213± 12.4

(n = 8)

Day 2

234± 6.8

(n = 9)

238± 9.4

(n = 10)

223± 9.9

(n = 10)

**214± 16.9

(n = 8)

Day 3

238± 6.2

(n = 9)

235± 7.4

(n = 10)

226± 9.3

(n = 10)

**216± 18.8

(n = 8)

Day 4

243± 8.2

(n = 9)

252± 7.7

(n = 10)

236± 11.4

(n = 10)

**223± 21.8

(n = 8)

 

 

Table 3: Fertility and breeding data

 

Control

10 mg/kg/d

30 mg/kg/d

75 mg/kd/d

Median precoital time for the first pairing period [d]

2

(n= 10)

3

(n= 10)

3

(n= 10)

2

(n= 9)

Median precoital time for the first pairing period [d]

 

 

 

1

(n= 1)

Gestation duration [d]

21.4± 0.53

(n= 9)

21.4± 0.52

(n= 10)

21.6± 0.52

(n= 10)

21.6± 0.53

(n= 9)

Corpora lutea

13.9± 1.3

(n= 9)

13.7± 1.3

(n= 10)

13.3± 1.9

(n= 10)

13.3± 2.0

(n= 9)

Implantation

13.4± 1.67

(n= 9)

13.5± 1.51

(n= 10)

11.4± 2.95

(n= 10)

12.2± 3.46

(n= 9)

Living pups total at first litter check

111

124

100

91

Dead pups total at first litter check

0

0

0

11

Mean living pups at first litter check

12.3± 1.66

(n= 9)

12.4± 2.12

(n= 10)

10.0± 3.13

(n= 10)

10.1± 5.44

(n= 9)

Living pups total day 4 post partum

111

124

100

84

Postnatal loss

0

0

0

7

Living pups day 4 post partum

12.3± 1.66

(n= 9)

12.4± 2.12

(n= 10)

10.0± 3.13

(n= 10)

9.3± 5.29

(n= 9)

Mean body weight of pups on day 1 post partum [g]

5.7± 0.43

(n= 9)

5.9± 0.63

(n= 10)

5.9± 0.74

(n= 10)

5.5± 0.83

(n= 8)

Mean body weight of pups on day 4 post partum [g]

7.9± 0.73

(n= 9)

8.3± 1.23

(n= 10)

8.4± 1.32

(n= 10)

7.1± 2.10

(n= 8)

 

 

Conclusions:
The reproduction toxicity of the registration substance was investigated according to the OECD 421.
NOAEL 30 mg/kg/d was obtained for the systemic toxicity of parental animals and NOAEL 30 mg(kg/d was obtained for the developmental toxicity.
Executive summary:

The reproduction toxicity of the registration substance was investigated according to the OECD 421.

The rats were treated with the registration substance daily per gavage at doses of 0, 10, 30 and 75 mg/kg/d. Males were treated two weeks prior to mating, and up to two weeks of mating phase and post mating phase. Females were treated two weeks prior to mating and up to the post partum day 4.

Reduced body weight gain was found for the animals treated at 75 mg/kg/d. No significant treatment related effect was found for the reproduction performance up to the dose of 75 mg/kg/d. The pups at 75 mg/kg/d exhibited reduced viability and reduced body weight.

NOAEL 30 mg/kg/d was obtained for the systemic toxicity of parental animals and NOAEL 30 mg(kg/d was obtained for the developmental toxicity.

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
The justification for the read-across provided in the attached document in Chapter 13: Assessment of the validity of the analogue approach
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: feed
Duration of treatment / exposure:
Exposure period: about 18 wk.
(F0 and F1) Premating exposure period (males): 10 wk.
Premating exposure period (females): 10 wk.
Duration of test: F0 pre-mating 10 wk, until F2 weaning.
Frequency of treatment:
Continuously
Dose / conc.:
0 mg/kg diet
Dose / conc.:
1 000 mg/kg diet
Dose / conc.:
2 000 mg/kg diet
Dose / conc.:
4 000 mg/kg diet
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Examination of F0 generation:
Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated
intervals. Males and females were paired for a 2-wk period, until mating was obtained. The F0 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded.
During lactation, the pups (F1 generation) were observed daily for survival and clinical signs; body weight was
recorded at designated intervals; the sex-ratio was recorded. On Day 4 post-partum, the size of each litter was
adjusted to obtain eight pups per litter (four males and four females). Reflex development was assessed at designated
time-points.
Sperm parameters (parental animals):
Epididymal and testicular sperm parameters were evaluated for both F0 and F1 males.
Litter observations:
Examination of F1 generation:
On Day 22 post-partum, one male and one female pup per litter were selected to constitute the F1 generation, which
comprised 25 males and 25 females per group. The F1 animals were observed daily for clinical signs and mortality. Body
weight and food consumption were recorded once a week. Sexual development of both males and females was assessed.
Neurobehavioural tests were conducted at designated intervals to assess auditory and visual functions. Spontaneous
locomotor activity was also evaluated when the animals were between 7 and 8 wk old. After sexual maturity, F1 male and F1 female animals were
paired. The F1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded.
During lactation, the pups (F2 generation) were observed daily for survival and clinical signs; body weight was recorded at designated intervals; the sex-ratio was recorded. On day 4 post-partum, the size of each litter was adjusted to obtain eight pups per litter (four males and four females).
Reflex development was assessed at designated time-points.
Postmortem examinations (parental animals):
Terminal examination of F0 and F1 animals:
After weaning of their respective progeny, F0 and F1 parent males and females were sacrificed. Designated organs were
weighed for F0 and F1 parents, as well as brain, spleen and thymus of one pup per sex per litter of each generation.
Epididymal and testicular sperm parameters were evaluated for both F0 and F1 males.
A macroscopic post-mortem examination was performed on all F0 and F1 parent males and females and on three pups per sex
and per litter of each F0 and F1 females killed at weaning. Any pups which died or were killed prematurely during the lactation period were also submitted for a macroscopic post-mortem examination.
Macroscopic lesions, reproductive organs, adrenals and pituitary glands were sampled in all parent animals. In all
pups, the macroscopic lesions were preserved. A microscopic examination was performed on macroscopic lesions,
reproductive organs, adrenals, and pituitary glands of all F0 and F1 parents of the control and high dose groups.
Particularly detailed histopathological examinations were performed for the ovaries and the testes.
Result: Not toxic to reproduction
F0 and F1 generations. At 4000 ppm (corresponding to approximately 123-208 mg/kg bw/d for F0 males and females and to 202-252 mg/kg bw/d for F1 males and females, respectively), a slightly to moderately lower mean food consumption and mean body weight gain were recorded during most of the dosing period in both parental males and females of the two generations and was associated with reduced liver weights. Necropsy of these animals (parents of both generations) revealed dilation of the cecum, colon or ileum in some animals (more marked in F0 parents). No effects were noted on sperm parameters or on histopathological examination of sexual organs. Slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of F1 parent females) and a delay in sexual development. Lower spleen weights were also noted for each progeny.
At 2000 ppm (corresponding to approximately 61-101 mg/kg bw/d for F0 males and females and 96-123 mg/kg bw/d for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. Necropsy of parents of both generations revealed dilatation of the cecum in some animals of the F0 generation and in a single animal of the F1 generation. This was associated with lower liver weights in parental animals of both generations. No effects were noted on parental fertility as assessed by normal mating, gestation and delivery and, particularly, there were no effects on sperm parameters or at histopathological examination of sexual organs. Except for a marginally lower spleen weight of the progeny of each generation, no other effects were noted on their development.
At 500 ppm (corresponding to approximately 16-25 mg/kg bw/d for F0 males and females and 24-31 mg/kg bw/d for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. This was associated in the F1 generation with lower liver weights of parental males and females. No effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny. As lower food consumption is known to occur due to palatability of compound. As no other substance related effects were seen than can be attributed to lower food intake, the level of 500 ppm test substance in the diet, corresponding to 16-25 mg/kg bw/d, should be regarded as NOAEL for parent and F1generation.

At 4000 ppm, number of implantation sites and litter size at birth were reduced. The progeny (F2) also showed lower pup weights. No effects were seen in F2 offspring at 2000 ppm regarding pup development and survival until weaning, and macroscopic examination after sacrifice at weaning. At the highest level of 4000 ppm pup weight gain was slightly lower during lactation, and upon necropsy dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females. Therefore, 4000 ppm can be regarded as LOAEL, and 2000 ppm as NOAEL for F2.
Key result
Dose descriptor:
NOAEL
Remarks:
general toxiicity
Effect level:
500 mg/kg diet
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: neoplastic
Remarks on result:
other: corresponds to 8-12.5 mg/kg/d ADBAC
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
2 000 mg/kg diet
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: corresponds to 30.5-50.5 mg/kg/d ADBAC
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
500 mg/kg diet
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Remarks on result:
other: corresponds to 12-15.5 mg/kg/d ADBAC
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction performance
Effect level:
2 000 mg/kg diet
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: corresponds to 48-61.5 mg/kg/d ADBAC
Result: Not toxic to reproduction
F0 and F1 generations. At 4000 ppm (corresponding to approximately 123-208 mg/kg bw/d for F0 males and females and to 202-252 mg/kg bw/d for F1 males and females, respectively), a slightly to moderately lower mean food consumption and mean body weight gain were recorded during most of the dosing period in both parental males and females of the two generations and was associated with reduced liver weights. Necropsy of these animals (parents of both generations) revealed dilation of the cecum, colon or ileum in some animals (more marked in F0 parents). No effects were noted on sperm parameters or on histopathological examination of sexual organs. Slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of F1 parent females) and a delay in sexual development. Lower spleen weights were also noted for each progeny. At 2000 ppm (corresponding to approximately 61-101 mg/kg bw/d for F0 males and females and 96-123 mg/kg bw/d for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. Necropsy of parents of both generations revealed dilatation of the cecum in some animals of the F0 generation and in a single animal of the F1 generation. This was associated with lower liver weights in parental animals of both generations. No effects were noted on parental fertility as assessed by normal mating, gestation and delivery and, particularly, there were no effects on sperm parameters or at histopathological examination of sexual organs. Except for a marginally lower spleen weight of the progeny of each generation, no other effects were noted on their development. At 500 ppm (corresponding to approximately 16-25 mg/kg bw/d for F0 males and females and 24-31 mg/kg bw/d for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. This was associated in the F1 generation with lower liver weights of parental males and females. No effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny. As lower food consumption is known to occur due to palatability of compound. As no other substance related effects were seen than can be attributed to lower food intake, the level of 500 ppm test substance in the diet, corresponding to 16-25 mg/kg bw/d, should be regarded as NOAEL for parent and F1generation. At 4000 ppm, number of implantation sites and litter size at birth were reduced. The progeny (F2) also showed lower pup weights. No effects were seen in F2 offspring at 2000 ppm regarding pup development and survival until weaning, and macroscopic examination after sacrifice at weaning. At the highest level of 4000 ppm pup weight gain was slightly lower during lactation, and upon necropsy dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females. Therefore, 4000 ppm can be regarded as LOAEL, and 2000 ppm as NOAEL for F2.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
2 000 mg/kg diet
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: corrresponds to 48-61.5 mg/kg/d ADBAC
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
2 000 mg/kg diet
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: corresponds to 48- 61.5 mg/kg/d ADBAC
Key result
Reproductive effects observed:
no

The mean achieved dosages of the test substance for the dose-levels of 500, 2000 and 4000 ppm of test substance were as follows:
F0 generation
- males (Days 1 to 106): 16, 61 and 123 mg/kg bw/d, respectively,
- females: during premating period (Days 1 to 71): 19, 74 and 154 mg/kg bw/d, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 18, 69 and 145 mg/kg bw/d, respectively,
    during lactation period (Days 1 to 21 p.p.): 37, 159 and 326 mg/kg bw/d, respectively.
F1 generation
- males (Days 1 to 120): 24, 96 and 202 mg/kg bw/d, respectively,
- females:
    during premating period (Days 1 to 64): 32, 127 and 269 mg/kg bw/d, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 21, 83 and 164 mg/kg bw/d, respectively,
    during lactation period (Days 1 to 21 p.p.): 41, 162 and 323 mg/kg bw/d, respectively.

The actual intake of test substance for both males and females given 500, 2000 and 4000 ppm throughout the study is approximately
16-25, 61-101 and 123-208 mg/kg bw/d, respectively  for the F0 generation and 24-31, 96-123 and 202-252 mg/kg bw/d for the F1 generation.

Conclusions:
The registration substance is expected to be of no significant reproduction toxicity based on the read-across to structurally related substance C12-16 ADBAC.
The reproduction toxicity of C12-16 ADBAC was investigated according to the OECD 416. No specific concern for the reproduction toxicity could be assinged to C12-16 ADBAC.
Executive summary:

The registration substance is expected to be of no significant reproduction toxicity based on the read-across to C12 -16 ADBAC.

In a two-generation study, C12-16 ADBAC was administered in the diet to male and female Sprague Dawley rats at 0, 500 (i.e.,16-25 and 24-31 mg/kg bw/day or 8-12.5 and 12-15.5 mg a.i./kg bw/day in males and females); 2,000 (i.e., 61-101and 96-123 mg/kg bw/day or 30.5-50 and 48 to 61.5 mg a.i./kg bw/day in males and females) or 4,000 ppm (i.e., 123-208 and 202-252 mg/kg bw/day or 61-104 and 101-126 mg a.i./kg bw/day in males and females), before and through mating and gestation until the end of the lactation period in both F0 and F1 generations.

At 2,000 ppm, F0 (males) and F1 parents showed marginally to slightly lower body weight gains and reduced food consumption. Necropsy of parents of both generations revealed dilatation of the caecum in some animals. This was associated with lower liver weights in parental animals of both generations.

At 4,000 ppm, in F0 and F1 parents, number of implantation sites and litter size at birth were reduced. The progeny (F2) also showed lower pup weights. Pup weight gain was slightly lower during lactation. Upon necropsy, dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females.

Treatment with the test substance had no effect on mating, fertility and behavioural parameters in F0 and F1 parental Sprague-Dawley rats at treatment levels up to 2,000 ppm. No effect was recorded on litter parameters and on pre- and post-natal development of either generation at 2,000 ppm.

The NOAEL for parental toxicity was 2000 ppm (equivalent to ca. 50 mg/kg/d),  the NOAEL for developmental toxicity was 2000 ppm (equivalent to ca- 50 mg/kg/d) and the NOAEL for fertility was 4000 ppm (equivalent to ca. 100 mg/kg/d). No specific concern for the reproduction toxicity could be assigned to C12 -16 ADBAC.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: feed
Duration of treatment / exposure:
Exposure period: about 18 wk.
(F0 and F1) Premating exposure period (males): 10 wk.
Premating exposure period (females): 10 wk.
Duration of test: F0 pre-mating 10 wk, until F2 weaning.
Frequency of treatment:
Continuously
Dose / conc.:
0 mg/kg diet
Dose / conc.:
500 mg/kg diet
Dose / conc.:
2 000 mg/kg diet
Dose / conc.:
4 000 mg/kg diet
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Examination of F0 generation:
Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated
intervals. Males and females were paired for a 2-wk period, until mating was obtained. The F0 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded.
During lactation, the pups (F1 generation) were observed daily for survival and clinical signs; body weight was
recorded at designated intervals; the sex-ratio was recorded. On Day 4 post-partum, the size of each litter was
adjusted to obtain eight pups per litter (four males and four females). Reflex development was assessed at designated
time-points.
Sperm parameters (parental animals):
Epididymal and testicular sperm parameters were evaluated for both F0 and F1 males.
Litter observations:
Examination of F1 generation:
On Day 22 post-partum, one male and one female pup per litter were selected to constitute the F1 generation, which
comprised 25 males and 25 females per group. The F1 animals were observed daily for clinical signs and mortality. Body
weight and food consumption were recorded once a week. Sexual development of both males and females was assessed.
Neurobehavioural tests were conducted at designated intervals to assess auditory and visual functions. Spontaneous
locomotor activity was also evaluated when the animals were between 7 and 8 wk old. After sexual maturity, F1 male and F1 female animals were
paired. The F1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded.
During lactation, the pups (F2 generation) were observed daily for survival and clinical signs; body weight was recorded at designated intervals; the sex-ratio was recorded. On day 4 post-partum, the size of each litter was adjusted to obtain eight pups per litter (four males and four females).
Reflex development was assessed at designated time-points.
Postmortem examinations (parental animals):
Terminal examination of F0 and F1 animals:
After weaning of their respective progeny, F0 and F1 parent males and females were sacrificed. Designated organs were
weighed for F0 and F1 parents, as well as brain, spleen and thymus of one pup per sex per litter of each generation.
Epididymal and testicular sperm parameters were evaluated for both F0 and F1 males.
A macroscopic post-mortem examination was performed on all F0 and F1 parent males and females and on three pups per sex
and per litter of each F0 and F1 females killed at weaning. Any pups which died or were killed prematurely during the lactation period were also submitted for a macroscopic post-mortem examination.
Macroscopic lesions, reproductive organs, adrenals and pituitary glands were sampled in all parent animals. In all
pups, the macroscopic lesions were preserved. A microscopic examination was performed on macroscopic lesions,
reproductive organs, adrenals, and pituitary glands of all F0 and F1 parents of the control and high dose groups.
Particularly detailed histopathological examinations were performed for the ovaries and the testes.
Result: Not toxic to reproduction
F0 and F1 generations. At 4000 ppm (corresponding to approximately 123-208 mg/kg bw/d for F0 males and females and to 202-252 mg/kg bw/d for F1 males and females, respectively), a slightly to moderately lower mean food consumption and mean body weight gain were recorded during most of the dosing period in both parental males and females of the two generations and was associated with reduced liver weights. Necropsy of these animals (parents of both generations) revealed dilation of the cecum, colon or ileum in some animals (more marked in F0 parents). No effects were noted on sperm parameters or on histopathological examination of sexual organs. Slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of F1 parent females) and a delay in sexual development. Lower spleen weights were also noted for each progeny.
At 2000 ppm (corresponding to approximately 61-101 mg/kg bw/d for F0 males and females and 96-123 mg/kg bw/d for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. Necropsy of parents of both generations revealed dilatation of the cecum in some animals of the F0 generation and in a single animal of the F1 generation. This was associated with lower liver weights in parental animals of both generations. No effects were noted on parental fertility as assessed by normal mating, gestation and delivery and, particularly, there were no effects on sperm parameters or at histopathological examination of sexual organs. Except for a marginally lower spleen weight of the progeny of each generation, no other effects were noted on their development.
At 500 ppm (corresponding to approximately 16-25 mg/kg bw/d for F0 males and females and 24-31 mg/kg bw/d for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. This was associated in the F1 generation with lower liver weights of parental males and females. No effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny. As lower food consumption is known to occur due to palatability of compound. As no other substance related effects were seen than can be attributed to lower food intake, the level of 500 ppm test substance in the diet, corresponding to 16-25 mg/kg bw/d, should be regarded as NOAEL for parent and F1generation.

At 4000 ppm, number of implantation sites and litter size at birth were reduced. The progeny (F2) also showed lower pup weights. No effects were seen in F2 offspring at 2000 ppm regarding pup development and survival until weaning, and macroscopic examination after sacrifice at weaning. At the highest level of 4000 ppm pup weight gain was slightly lower during lactation, and upon necropsy dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females. Therefore, 4000 ppm can be regarded as LOAEL, and 2000 ppm as NOAEL for F2.
Key result
Dose descriptor:
NOAEL
Remarks:
general toxiicity
Effect level:
500 mg/kg diet
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
4 000 mg/kg diet
Sex:
male/female
Basis for effect level:
other: no significant effect up to the highest dose
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
2 000 mg/kg diet
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
4 000 mg/kg diet
Sex:
male/female
Basis for effect level:
other: no significant effect up to the highest dose
Result: Not toxic to reproduction
F0 and F1 generations. At 4000 ppm (corresponding to approximately 123-208 mg/kg bw/d for F0 males and females and to 202-252 mg/kg bw/d for F1 males and females, respectively), a slightly to moderately lower mean food consumption and mean body weight gain were recorded during most of the dosing period in both parental males and females of the two generations and was associated with reduced liver weights. Necropsy of these animals (parents of both generations) revealed dilation of the cecum, colon or ileum in some animals (more marked in F0 parents). No effects were noted on sperm parameters or on histopathological examination of sexual organs. Slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of F1 parent females) and a delay in sexual development. Lower spleen weights were also noted for each progeny. At 2000 ppm (corresponding to approximately 61-101 mg/kg bw/d for F0 males and females and 96-123 mg/kg bw/d for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. Necropsy of parents of both generations revealed dilatation of the cecum in some animals of the F0 generation and in a single animal of the F1 generation. This was associated with lower liver weights in parental animals of both generations. No effects were noted on parental fertility as assessed by normal mating, gestation and delivery and, particularly, there were no effects on sperm parameters or at histopathological examination of sexual organs. Except for a marginally lower spleen weight of the progeny of each generation, no other effects were noted on their development. At 500 ppm (corresponding to approximately 16-25 mg/kg bw/d for F0 males and females and 24-31 mg/kg bw/d for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. This was associated in the F1 generation with lower liver weights of parental males and females. No effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny. As lower food consumption is known to occur due to palatability of compound. As no other substance related effects were seen than can be attributed to lower food intake, the level of 500 ppm test substance in the diet, corresponding to 16-25 mg/kg bw/d, should be regarded as NOAEL for parent and F1generation. At 4000 ppm, number of implantation sites and litter size at birth were reduced. The progeny (F2) also showed lower pup weights. No effects were seen in F2 offspring at 2000 ppm regarding pup development and survival until weaning, and macroscopic examination after sacrifice at weaning. At the highest level of 4000 ppm pup weight gain was slightly lower during lactation, and upon necropsy dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females. Therefore, 4000 ppm can be regarded as LOAEL, and 2000 ppm as NOAEL for F2.
Key result
Dose descriptor:
NOAEL
Remarks:
offspring
Generation:
F1
Effect level:
2 000 mg/kg diet
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
offspring
Generation:
F2
Effect level:
2 000 mg/kg diet
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Reproductive effects observed:
no

The mean achieved dosages of the test substance for the dose-levels of 500, 2000 and 4000 ppm of test substance were as follows:
F0 generation
- males (Days 1 to 106): 16, 61 and 123 mg/kg bw/d, respectively,
- females: during premating period (Days 1 to 71): 19, 74 and 154 mg/kg bw/d, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 18, 69 and 145 mg/kg bw/d, respectively,
    during lactation period (Days 1 to 21 p.p.): 37, 159 and 326 mg/kg bw/d, respectively.
F1 generation
- males (Days 1 to 120): 24, 96 and 202 mg/kg bw/d, respectively,
- females:
    during premating period (Days 1 to 64): 32, 127 and 269 mg/kg bw/d, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 21, 83 and 164 mg/kg bw/d, respectively,
    during lactation period (Days 1 to 21 p.p.): 41, 162 and 323 mg/kg bw/d, respectively.

The actual intake of test substance for both males and females given 500, 2000 and 4000 ppm throughout the study is approximately
16-25, 61-101 and 123-208 mg/kg bw/d, respectively  for the F0 generation and 24-31, 96-123 and 202-252 mg/kg bw/d for the F1 generation.

The actual F0 intake of C12 -C16 ADCBA throughout the study was 8, 30.5 and 61.5 mg/kg/d for males and 9 -18.5, 34.5 -79.5 and 72.5 -163 mg/kg/d for females.

And the actual F1 intake of C12 -C16 ADCBA throughout the study was 12, 48 and 101 mg/kg/d for males and 10.5 -20.5, 41.5 -81 and 82 -161.5 mg/kg/d for females.

Conclusions:
The reproduction toxicity of C12-16 ADBAC was investigated according to the OECD 416 via feeting. The NOAEL for parental toxicity was 2000 ppm (equivalent to ca. 50 mg/kg/d), the NOAEL for developmental toxicity was 2000 ppm (equivalent to ca- 50 mg/kg/d) and the NOAEL for fertility was 4000 ppm (equivalent to ca. 100 mg/kg/d). No specific concern for the reproduction toxicity could be assigned.
Executive summary:

In a two-generation study, C12-16 ADBAC (50% a.i.) was administered in the diet to male and female Sprague Dawley rats at 0, 500, 2,000 or 4,000 ppm. The F0 generation animals were treated 10 weeks prior to mating, through mating and gestation and until the end of the lactation period. The F1 generation animals were treated throughly until weaning of their offspring.

The actual F0 intake of C12 -C16 ADCBA throughout the study was 8, 30.5 and 61.5 mg/kg/d for males and 9 -18.5, 34.5 -79.5 and 72.5 -163 mg/kg/d for females. And the actual F1 intake of C12 -C16 ADCBA throughout the study was 12, 48 and 101 mg/kg/d for males and 10.5 -20.5, 41.5 -81 and 82 -161.5 mg/kg/d for females.

At 2,000 ppm, F0 (males) and F1 parents showed marginally to slightly lower body weight gains and reduced food consumption. Necropsy of parents of both generations revealed dilatation of the caecum in some animals.

At 4,000 ppm, in F0 and F1 parents, number of implantation sites and litter size at birth were reduced. The progeny (F2) also showed lower pup weights. Pup weight gain was slightly lower during lactation. Upon necropsy, dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females.

Treatment with the test substance had no effect on mating, fertility and behavioural parameters in F0 and F1 parental Sprague-Dawley rats at treatment levels up to 2000 ppm. No effect was recorded on litter parameters and on pre- and post-natal development of either generation at 2,000 ppm.

The NOAEL for parental toxicity was 2000 ppm (equivalent to ca. 50 mg/kg/d),  the NOAEL for developmental toxicity was 2000 ppm (equivalent to ca- 50 mg/kg/d) and the NOAEL for fertility was 4000 ppm (equivalent to ca. 100 mg/kg/d).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Species:
rat
Quality of whole database:
Data-set reliable and sufficient concerning classification & labelling requirements.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

For the assessment of the developmental toxicity of the registration substance two key studies are to be used: one developmental toxicity study in rats (similar to OECD 414) and one developmental toxicity study in rabbits (similar to OECD 414) on the read-across source substanceC

C12-16 ADBAC was administered to groups of 25 pregnant rats by gavage at dose levels of 0, 10, 30 and 100 mg/kg bw/day, once daily from Day 6 to Day 15 of gestation inclusive. Control animals were treated with the vehicle (Milli-Q water). Clinical observations were made twice daily and maternal body weights were measured on Gestation Days 0, 6, 9, 12, 15, 18 and 21. Maternal food consumption was measured at 3 day intervals from Day 0 to Day 21. All surviving females were sacrificed on Day 21 and the foetuses were examined for visceral and skeletal variations and malformations. No mortality was observed during the study. Treatment-related clinical signs included perioral wetness and audible respiration in the high dose group. Audible respiration was also observed in the mid dose group. Food consumption was reduced between Days 6 to 9 in the mid and high dose groups. There were no effects of treatment on gestational body weight and body weight gain and gravid uterine weight at any dose. There were also no treatment-related differences in gestational parameters including total number of implantations, number of viable and non-viable implants between the control and the test groups. Further, there were no effects of treatment on fetal body weights per litter, or on the incidences of external, visceral and skeletal malformations and variations. Based on the results of this study, C12-16 ADBAC was found to produce some adverse effects in pregnant rats at 30 and 100 mg/kg bw/day; however, no developmental toxicity, including teratogenicity was observed at any of the dosages. The NOAEL for maternal toxicity was 10 mg/kg bw/d (i.e., equivalent to 8.1 mg a.i./kg bw/d) and the NOAEL for developmental toxicity was 100 mg/kg bw/d (i.e., equivalent to 81 mg a.i./kg bw/d).

C12-16 ADBAC was administered to pregnant rabbits by gavage from Day 6 to 28 post-coitum at the dose-levels of 3, 10 or 30 mg/kg bw/day of active substance. The dose of 30 mg/kg bw/day caused the death of three females, severe clinical condition or abortion in two other females and transient, lower maternal body weight gain. Necropsies revealed in 8/22 females accentuated lobular patterns in the liver, whitish areas and/or blackish deposits in the stomach mucosa and dilated intestines. At 10 mg/kg bw/day, relevant necropsy findings were noted in 5/22 females (dilated gall bladder, accentuated lobular pattern, pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa). No maternal toxicity or effects on litter data parameters or embryo-foetal development were noted at 3 mg/kg bw/day. Under the condition of the study, the NOAEL for maternal toxicity was 3 mg/kg bw/day while the NOAEL for embryo-foetal development was 30 mg/kg bw/day.

In both studies the obtained NOAELs for developmental toxicity corresponded to the highest dose tested. No significant developmental toxicity was found for C12 -16 ADBAC. Likewise no significant developmental toxicity can be assigned to the registration substamce.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
The justification for the read-across provided in the attached document in Chapter 13: Assessment of the validity of the analogue approach
Reason / purpose for cross-reference:
read-across source
Species:
rabbit
Key result
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day
Basis for effect level:
body weight and weight gain
gross pathology
mortality
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Basis for effect level:
other: no effect up to the highest dose
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
The registration substance is expected to be of no significant developmental toxicity based on the read-across to C12 -16 ADBAC.
The developmental toxicity of C12-16 ADBAC was investigated  in accordance with OECD 414 in rabbits . The maternal NOAEL was 3 mg/kg/d and the developmental NOAEL was 30 mg/kg/d. No indication of developmental toxicity was found.
Executive summary:

The registration substance is expected to be of no significant developmental toxicity based on the read-across to C12 -16 ADBAC.

The developmental toxicity of C12-16 ADBAC was investigated  in accordance with OECD 414 in rabbits . The maternal NOAEL was 3 mg/kg/d and the developmental NOAEL was 30 mg/kg/d. No indication of developmental toxicity was found.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
The justification for the read-across provided in the attached document in Chapter 13: Assessment of the validity of the analogue approach
Reason / purpose for cross-reference:
read-across source
Species:
rat
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Basis for effect level:
clinical signs
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Basis for effect level:
other: no effect up to the highest dose
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
The registration substance is expected to be of no significant developmental toxicity based on the read-across to C12 -16 ADBAC.
The developmental toxicity of C12-16 ADBAC was investigated in accordance with OECD 414 . The maternal NOAEL was 10 mg/kg/d and the developmental NOAEL was 100 mg/kg/d. No indication of developmental toxicity was found.
Executive summary:

The registration substance is expected to be of no significant developmental toxicity based on the read-across to C12 -16 ADBAC.

The developmental toxicity of C12-16 ADBAC was investigated  in accordance with OECD 414 . The maternal NOAEL was 10 mg/kg/d and the developmental NOAEL was 100 mg/kg/d. No indication of developmental toxicity was found.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Test substance was administered from Days 6-15 of gestation
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Test substance was administered from Days 6-15 of gestation
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Sprague Dawley CD rats (Crl: CD BR) were obtained from Charles River Breeding Laboratories, Portage MI USA.
- Age at study initiation: 11 weeks
- Weight at study initiation: 245.0-246.6 g
- Housing: Animals were housed singly in stainless steel cage, wire mesh caging; dimension, 22.5 x 15.5 x 18.0 cm during the study.
- Diet: Ground Certified Rodent Chow # 5002 (Ralston Purina Company, St. Louis, MO), ad libitum
- Water: Tap water, ad libitum. Water was provided by an automatic watering system with demand control valves mounted on each rack.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 66-77°F
- Humidity: 40-70%
- Air changes: 8/h

IN-LIFE DATES: From: 14 October 1991 To: 29 November 1991
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(Milli-Q water)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared by dissolving appropriate amount of the test substance with Milli-Q water. Concentrations were adjusted for percent active ingredient of the test substance.
- Rate of dose preparation: Dosing solutions were prepared once.
- Storage of dose formulations: Room temperature

VEHICLE
- Concentration in vehicle: 0, 2, 6, and 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/d

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analysed for concentration, homogeneity and stability by HPLC method.
- The homogeneity study performed on samples from the 10 and 100 mg/kg bw/d concentrations indicated that the test substance was uniformly distributed in the solution.
- The stability study conducted on lowest and highest concentrations indicated that the test substance was stable in the solutions for at least 14 d in a glass flask when stored at room temperature.
- Concentration verification analyses of the dosing solutions showed analytical mean values ranging from 94.5 to 104.8% of nominal for all 3 concentrations.
Details on mating procedure:
- Impregnation procedure: Co-housed
- If cohoused: Animals were co-housed in stainless steel wire mesh cages (30.5 x 31.0 x 18.0 cm).
- M/F ratio per cage: 1:1
- Length of cohabitation: Animals were cohoused until evidence of copulation was observed
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: Each male was paired only once in the study.
- Verification of same strain and source of both sexes: Male and female rats were obtained from same source
- Proof of pregnancy: Presence of vaginal copulation plug. This day was designated as Day 0 of gestation
Duration of treatment / exposure:
From Day 6-15 of gestation.
Frequency of treatment:
Once daily during exposure period
Duration of test:
16 d (From Day 6 to Day 21 post coitum)
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosages were selected by the study sponsor on the basis of a range finding study with the test substance (BBRC Project Report 54-613).
- Rationale for animal assignment: Immediately after mating the female rats were randomly allocated to treatment groups and control group using a stratified randomization program based on body weight on gestation Day 0.
- Animal assignment: Mated rats were assigned to the following groups.
Group 1 (vehicle control): 0 mg/kg bw/d
Group 2 (Low dose): 10 mg/kg bw/d
Group 3 (Mid dose): 30 mg/kg bw/d
Group 4 (High dose): 100 mg/kg bw/d
Maternal examinations:
Mortality: Yes
Time schedule: Twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Days 0, 6, 9, 12, 15, 18 and 21.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was recorded for every 3 d intervals from Days 0 to 21 post coitum (Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21)

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on: Day 21 post coitum
- The females were killed by CO2 asphyxiation and the fetuses removed by caesarean section.
- Organs examined: Gravid uterus, ovaries, cervix, vagina, and peritoneal and thoracic cavities

OTHER: Liver and gravid uterine were weighed.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- The fetuses were removed from the uterus, sexed, weighed individually and examined for variations and malformations including cleft palate
- External examinations: Yes (all per litter)
- Soft tissue examinations: Yes (approximately half per litter)
- Skeletal examinations: Yes (approximately half per litter)
- Head examinations: Yes (approximately half per litter)
- Body weight: Yes
Statistics:
- Levene’s test for equal variances, analysis of variance, and a pooled t-tests for pairwise comparisons.
- Nonparametric data were analyzed with Kruskal-Wallis test followed by Mann-Whitney U test when appropriate.
- Incidence data were compared using Fisher’s Exact Test.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY: All animals survived until scheduled necropsy.

CLINICAL SIGNS: Perioral wetness in 67% of dams and audible respiration in 3 dams of high dose group. One dam in this group exhibited dehydration, unkempt appearance, loose feces, urine stains, and perioral wetness. Audible respiration in 2 dams of mid dose group. One of these dams also exhibited urine stains, gasping, perinasal encrustation, loose feces and perioral wetness.

BODY WEIGHT: There were no effects of treatment on gestational body weight and body weight gain, and gravid uterine weight in any dose group.

FOOD CONSUMPTION: Food consumption was reduced between Days 6 to 9 in the mid and high dose group.

NECROPSY FINDINGS: Ulceration of stomach and gas-filled intestines, color changes in liver and lymph nodes and small spleen was seen in one dam of high dose group. Swollen liver was observed in one dam of mid dose group.

REPRODUCTION DATA: There were no treatment-related differences in the number of ovarian corpora lutea and in gestational parameters including total number of implantations, number of viable and nonviable implants.


Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
food consumption and compound intake
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
BODY WEIGHTS: No treatment-related effects on fetal body weights were observed in any group.

EXTERNAL EXAMINATION: No test substance-related relevant effects during the external examination of fetuses were noted in any group.

SKELETAL EXAMINATION: No treatment-related variations or malformations.

VISCERAL EXAMINATION: No treatment-related variations or malformations.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effect up to the highest dose
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
The developmental toxicity of C12-16 ADBAC was investigated in accordance with OECD 414 in rats . The maternal NOAEL was 10 mg/kg/d and the developmental NOAEL was 100 mg/kg/d. No indication of developmental toxicity was found.
Executive summary:
C12-16 ADBAC was administered to groups of 25 pregnant rats by gavage at dose levels of 0, 10, 30 and 100 mg/kg bw/day, once daily from Day 6 to Day 15 of gestation inclusive. Control animals were treated with the vehicle (Milli-Q water). Clinical observations were made twice daily and maternal body weights were measured on Gestation Days 0, 6, 9, 12, 15, 18 and 21. Maternal food consumption was measured at 3 day intervals from Day 0 to Day 21. All surviving females were sacrificed on Day 21 and the foetuses were examined for visceral and skeletal variations and malformations. No mortality was observed during the study. Treatment-related clinical signs included perioral wetness and audible respiration in the high dose group. Audible respiration was also observed in the mid dose group. Food consumption was reduced between Days 6 to 9 in the mid and high dose groups. There were no effects of treatment on gestational body weight and body weight gain and gravid uterine weight at any dose. There were also no treatment-related differences in gestational parameters including total number of implantations, number of viable and non-viable implants between the control and the test groups. Further, there were no effects of treatment on fetal body weights per litter, or on the incidences of external, visceral and skeletal malformations and variations. Based on the results of this study, C12-16 ADBAC was found to produce some adverse effects in pregnant rats at 30 and 100 mg/kg bw/day; however, no developmental toxicity, including teratogenicity was observed at any of the dosages. The NOAEL for maternal toxicity was 10 mg/kg bw/d (i.e., equivalent to 8.1 mg a.i./kg bw/d) and the NOAEL for developmental toxicity was 100 mg/kg bw/d (i.e., equivalent to 81 mg a.i./kg bw/d).
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
batch number is not presented for the vehicle (purified water) in the study report; autolysed foetuses were not sexed at the time of hysterectomy and head sections were archived in the same way as brain sections.
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Route of administration:
oral: gavage
Duration of treatment / exposure:
Day 6 to 28 post coitum
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
3 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
No. of animals per sex per dose:
22 mated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: GD 6-28
Key result
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day
Basis for effect level:
body weight and weight gain
gross pathology
mortality
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Basis for effect level:
other: no treatment related effect up to the highest dose
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Result: The test substance is not teratogenic in rabbits.

Maternal effects:
At 30 mg/kg bw/d:
- three females died and two females were prematurely sacrificed for ethical reasons or abortion,
- the relevant clinical signs concerned the deceased females and two prematurely sacrificed females. No other clinical signs were noted in females from this group.
- body weight gain was transiently reduced (GD 9-12, -70% below the control, p<0.05) but returned to normal values thereafter,
- the necropsies revealed in 8/22 females: accentuated lobular pattern in the liver, pale liver, whitish areas and/or blackish deposits and/or edema in the stomach mucosa, reddish or brownish foci on the lungs, blackish contents in
the intestines, dilated intestines and dilated gall bladder,

At 10 mg/kg bw/d:
- there were no deaths. Reduction of maternal body weight gain and food consumption did not reach statistical significance,

- There were no relevant clinical signs except for two females with blood in the bedding on Days 22 and 23 post-coitum or absence of feces from Day 26 post-coitum
- the necropsies revealed in 5/22 females: dilated gall bladder, accentuated lobular pattern, 

- pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa,

At 3 mg/kg bw/d:
no signs of maternal toxicity,

Teratogenic / embryotoxic effects:
There were no effects on litter data parameters and no treatment-related findings upon external, visceral or skeletal observation in any of the dose groups.

Litter and foetal evaluation:
The fluctuations noted in the mean number of corpora lutea, implantation sites and post-implantation were slight and not
dose-related, they were consequently considered not to be treatment-related.
The fluctuations recorded for the mean number of resorptions (early or late) and the mean number of dead foetuses and
consequently for the percentages of post-implantation loss were also not considered to be treatment-related, as they
were minimal and not dose-related. The percentage of male foetuses was considered similar among the groups and the fetal body weight was unaffected by treatment.

External, soft tissue and skeletal observations in foetuses and final fetal assessment:
The occurrence of some external and soft tissue malformations throughout all treated groups, including the
controls, did not suggest any substance-related origin because of their low incidence, absence of dose-relationship
and/or statistical significance.
Concerning the skeletal observations, no relevant malformations was noted but the presence of a full supernumerary 13th pair of ribs (as a foetal variation), was markedly increased at 10 and 30 mg/kg bw/day. These differences
in foetal or litter incidence, which were within background data, were most probably due to a low control value. The
incidence of one other skeletal variation (unossified 5th sternebra) was significantly greater but in the low dose-group only.

Conclusion: Test substance is not teratogenic in rabbits.

LO(A)EL maternal toxic effects:
10 mg/kg bw/d, based on necropsy findings in 5/22 animals.
Incidence was increased to 8/22 at 30 mg/kg bw/d (dilated gallbladder 3/22, accentuated lobular pattern liver 3/22).
Foci (reddish/brownish) in the lung was also observed in 2 females, but also in view of findings in range finding study and
parallel study with comparable compound, this can be caused by inadvertent presence of substance into the airways and
not attributable to systemic toxicity. Incidence was not increased in the top-dose group. There is an indication of
lower body weight gain, correlating to a lower food consumption, but that was not statistical significant and in
the high-dose goup not different from the mid-dose group.
Blackish content in stomack and intestines is indicative of local corrosive effects of test substance.


NO(A)EL maternal toxic effects:
3 mg/kg bw/d. There seems to be a dose-response related increase in necropsy findings in stomach, intestine and liver.
Dilated gallbladder incidence was not increased in highest dose group compared to mid-dose.
LO(A)EL embryotoxic / teratogenic effects:
Based on the number of foetuses presenting malformations or variations and in the absence of a treatment-related
increase of such observation, the embryo-fetal development was not considered to be affected by treatment.
NO(A)EL embryotoxic / teratogenic effects:
30 mg/kg bw/d, being the highest tested dose.

Conclusions:
The developmental toxicity of C12-16 ADBAC was investigated in accordance with OECD 414 in rabbits . The maternal NOAEL was 3 mg/kg/d and the developmental NOAEL was 30 mg/kg/d. No indication of developmental toxicity was found.
Executive summary:

A guideline equivalent developmental toxicity study was conducted in rabbits. C12-16 ADBAC was administered to pregnant rabbits by gavage from Day 6 to 28 post-coitum at the dose-levels of 3, 10 or 30 mg/kg bw/day of active substance. The dose of 30 mg/kg bw/day caused the death of three females, severe clinical condition or abortion in two other females and transient, lower maternal body weight gain. Necropsies revealed in 8/22 females accentuated lobular patterns in the liver, whitish areas and/or blackish deposits in the stomach mucosa and dilated intestines. At 10 mg/kg bw/day, relevant necropsy findings were noted in 5/22 females (dilated gall bladder, accentuated lobular pattern, pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa). No maternal toxicity or effects on litter data parameters or embryo-foetal development were noted at 3 mg/kg bw/day. Under the condition of the study, the NOAEL for maternal toxicity was 3 mg/kg bw/day while the NOAEL for embryo-foetal development was 30 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Species:
rabbit
Quality of whole database:
Data-set reliable and sufficient concerning classification & labelling requirements.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

No classification with respect to the reproduction toxicity is warranted for the registration substance according to the criteria of the EU Classification Labeling and Packaging Regulation (1272/2008/EC).

The results obtained in the reproduction toxicity screening study indicate absence of reproduction toxicity up to the dose associated with apparent maternal toxcity. In addition, no significant reproduction toxicity was found in studies (2 -generation study in rats, developmental toxicity studies in rats and rabbits) on the structurally related substance.

Additional information