Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The available two-generation reproductive toxicity studies with the read across substance in rats, indicate no concern for reproductive toxicity for the test substance. 

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Oct, 2003 - 21 May, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Deficiencies: Yes F0 animals: the day 11 samples taken for stability in closed bags and open feeders, were actually sampled on day 12 by error, males D26143 and D26154 given 1500 ppm were weighed again on day 17 due to aberrant values on day 15, for females D26364, pup No. 5 and D26393, pup No. 10 (given respectively 500 and 1500 ppm), macroscopic examination was performed although not scheduled in the study plan (data not presented in the study report), Male D26146 (given 1500 ppm): left testis and epididymis were mislaid before microscopic examination. F1 animals: according to the Study plan, the dosing period was 20 weeks. However, the study was finished in week 18, the dietary admixtures were analyzed in week 18 rather than week 20, as specified in the Study plan, because the study finished early, female D26509 given 4000 ppm and females D26439 and D26446 given 0 ppm were mated one extra day by error (the females had been positive for mating the previous day), during the mating periods (F0 and F1 animals), the food consumption was not recorded (typing error in the final study plan), organ weights with aberrant values have been excluded from mean calculation: thyroid glands of male D26210 (given 500 ppm), adrenal glands of male D26232 (given 1500 ppm) and pituitary gland of male D26234 (given 1500 ppm), male D26226 (given 500 ppm): dilated renal pelvis was observed for left kidney at macroscopic examination but no microscopic examination was performed, male D26262 (given 4000 ppm): adrenal glands were not found for the preparation of slides, female D26457 (given 500 ppm): pituitary gland was mislaid after weighing, female D26493, pup No. 4 (given 1500 ppm) found dead on day 2 post-partum was mislaid before macroscopic examination. These deviations were considered not to have compromised the validity or integrity of the study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Rat
Strain: Sprague-Dawley, Crl CD® (SD) IGS BR
Source: Charles River Laboratories France, L’Arbresle, France.
Sex: 100 males and 100 females
Age/weight at study initiation: At the beginning of the treatment period, the animals were approximately 5 to 6 weeks old and had a mean body
weight of 137 g (range: 104 g to 168 g) for the males and 118 g (range: 96 g to 139 g) for the females.
Number of animals per group: 25 males and 25 females
Duration of mating: 2 weeks
Deviations from standard protocol: Female D26509 given 4000 ppm and females D26439 and D26446 given 0 ppm were mated one extra day
by error (the females had been positive for mating the previous day).
Control animal: Yes

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Administration/ Exposure: Oral
Duration of exposure before mating: 10 weeks
Duration of exposure in general P, F1, F2 males, females From beginning of the study until sacrifice of Parent, F1, F2-generation
Type: oral In food
Concentration Food consumption per day: 0, 500, 1500 and 4000 ppm ad libitum, for groups 1, 2, 3 and 4, respectively.
Vehicle: The test item was mixed in the powdered maintenance diet UAR A04C P2.5 (UAR, Villemoisson, Epinay-sur-Orge, France).
Controls: Vehicle

Details on mating procedure:

Males and females were paired for, at most, a 2-week period, until mating was obtained.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Doses were verified for test substance concentrations in regular intervals during the study.

Duration of treatment / exposure:

Exposure: Premating exposure period (males): 10 weeks
Premating exposure period (females): 10 weeks
Duration of test: F0 pre-mating 10 weeks, until F2 weaning. (Continuously)

Frequency of treatment:

Continuously

Details on study schedule:

F0 generation: 10 weeks before mating, during the mating period until sacrifice (weaning of pups) for the males, 10 weeks before mating, during the mating, pregnancy and lactation periods (until day 21 post-partum) for the females.

A group of 25 males and 25 females received untreated diet alone, under the same experimental conditions, and acted as a reference control group. 

F1 generation: At weaning of the F1 generation, on day 22 post-partum, three groups of 25 male and 25 female Sprague-Dawley rats received the same test item, under the same experimental conditions as above, during their growth, adulthood, mating, pregnancy and lactation, until weaning of the pups (F2 generation).

A group of 25 males and 25 females received untreated diet alone under the same experimental conditions and acted as a reference control group. 

Remarks:

Doses / Concentrations:
500, 1500, and 4000 ppm of test substance (40% active DDAC) (i.e., corresponding to 203, 608 and 1620 ppm of DDAC)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
14, 39 and 109 mg a.i./kg bw/day for males and 18, 51 and 137 mg a.i./kg bw/day in females
Basis:
nominal in diet

No. of animals per sex per dose:

25 per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

Refer to study design above

Positive control:

Not applicable

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, at most, a 2-week period, until mating was obtained.  The F0 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded. The F0 parent males and females were sacrificed after weaning of their progeny. During lactation, the pups from the F1 generation were observed daily for survival and clinical signs. Body weight was measured at designated intervals and 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 endpoints.

Oestrous cyclicity (parental animals):

Refer to study design

Sperm parameters (parental animals):

Refer to study design

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. Neurobehavioral tests were conducted at designated intervals to assess auditory and visual function. Spontaneous locomotor activity was also evaluated twice over a 60-min interval when the animals were 7 and 8 weeks old. When the animals were 12 weeks old, F1 males and F1 females were paired.
The F1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded. During lactation, the pups from 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 endpoints.

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 in 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 female killed at weaning.  Any pups which died or were killed prematurely during the lactation period were also submitted for 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. A detailed histopathological examination was performed on the ovaries and the testes.

Postmortem examinations (offspring):

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 in 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 female killed at weaning.  Any pups which died or were killed prematurely during the lactation period were also submitted for 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. A detailed histopathological examination was performed on the ovaries and the testes.

Statistics:

No data

Reproductive indices:

Please refer to study design for F0 animals

Offspring viability indices:

Please refer to study design for F1 animals

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

F0 generation
Test group 4 (4000 ppm): - no treatment-related deaths and no remarkable clinical signs,  - for the males, a lower body weight gain during the premating period (days 1-15), and food consumption was affected throughout the study,  - for the females, a lower body weight gain was noted during the first week of the premating period, during the gestation and the lactation, and food consumption was affected from week 2 of the study, but no effect was detected during the lactation, - there were no apparent effects on mating, fertility, fecundity or delivery,  - the pup body weight gain was significantly lower than controls from day 4 p.n.,  - no disturbances of the reflex development in the pups,  - none of the seminology parameters evaluated in F0 parents males were affected,  - distension of cecum with feces was seen at the autopsy in 13/25 F0 males, with no histopathological findings in this organ,  - higher adrenal weights (p<0.01) in F0 females correlated with enlargement of the gland and cortical cell hypertrophy seen at histopathology, - lower spleen weights in male and female pups sacrificed at weaning (p<0.01),  - no treatment-related findings were observed in male and female genital organs, and the accessory organs.          
Test group 3 (1500 ppm): - no treatment-related deaths, - no remarkable clinical signs, - neither body weight gain nor food consumption of parent animals were affected, - there were no apparent effects on mating, fertility, fecundity or delivery, - no effect on the pup body weight gain, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F0 parents males were affected, - no treatment-related differences in organ weights were recorded, - neither macroscopic nor microscopic findings were observed.
Test group 2 (500 ppm): - no treatment-related deaths, - no remarkable clinical signs, - neither body weight gain nor food consumption of parent animals were affected, - there were no apparent effects on mating, fertility, fecundity or delivery, - no effect on the pup body weight gain, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F0 parents males were affected, - no treatment-related differences in organ weights were recorded, - neither macroscopic nor microscopic findings were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

F1 generation
Test group 4 (4000 ppm): - no treatment-related deaths, - no remarkable clinical signs, - for the males, there was a lower body weight gain until day 43 of the premating period and food consumption was affected, mainly during the same period of the study, - for the females, a lower body weight gain was noted during the first 2 weeks of the premating period, from GD 7 (GD: gestation day) and during the lactation. The food consumption was affected during the premating period, during GD 0-14 and during days 7 14 p.p., - there were no apparent effects on mating, fertility, fecundity or the progress of delivery,  - the pup body weight gain was significantly lower than controls during the lactation, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F1 parents males were affected, - none of the organ weight differences recorded in F1 parents were directly treatment-related, - lower spleen weights were recorded in F1 pups, - no relevant findings were seen at necropsy of parents or pups, - neither macroscopic nor microscopic findings were observed.
Test group 3 (1500 ppm): - no treatment-related deaths, - no remarkable clinical signs, - neither body weight gain nor food consumption of parent animals were affected, - there were no apparent effects on mating, fertility, fecundity or the progress of delivery, - increase in the male ratio, - no effect on the pup body weight gain, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F1 parents males were affected. - no treatment-related differences in organ weights were recorded, - neither macroscopic nor microscopic findings were observed.
Test group 2 (500 ppm): - no treatment-related deaths, - no remarkable clinical signs, - neither body weight gain nor food consumption of parent animals were affected, - there were no apparent effects on mating, fertility, fecundity or the progress of delivery, - increase in the male ratio, - no effect on the pup body weight gain, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F1 parents males were affected. - no treatment-related differences in organ weights were recorded, - neither macroscopic nor microscopic findings were observed.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects on mating behaviour, fertility and gestation, of each generation and for development, growth and survival of each progeny

Key result

Reproductive effects observed:

no

Result: Not toxic to reproduction
concentrations of 500, 1500 or 4000 ppm test substance,
corresponding to 203, 608 or 1620 ppm of DDAC.

The estimation of the achieved dosages of test item
(expressed as mg of DDAC/kg/day) are summarized in the
following table:
Mean achieved dosages (in mg DDAC/kg/day)
Concentration (ppm)         500         1500         4000
F0 generation
Males
. premating (days 1-71)         17         49         137
. post-mating (days 85-120)     10         30         84
Females
. premating (days 1-71)         20         58         157
. gestation (GD 0-20)          15         45         52
. lactation (days 1-21 p.n.)    31         93         261
F1 generation
Males
. premating (days 1-64)         22         65         183
. post-mating (days 85-120)     10         31         94
Females
. premating (days 1-64)         23         69         198
. gestation (GD 0-20)          17         48         57
. lactation (days 1-21 p.n.)    36         93         263

Conclusions:

Under the study conditions, the NOAEL for parental toxicity was 1500 ppm (39-51 mg a.i./kg bw/day) for the male and female animals. The NOAEL for mating behaviour, fertility and gestation of each generation and for development, growth and survival of each progeny was 4000 ppm (109-137 mg a.i./kg bw/day).

Executive summary:

A two-generation study was conducted to determine the toxicity to reproduction of the test substance, DDAC (40% active in water) according to OECD Guideline 416, in compliance with GLP. In this study, the substance was administered in the diet to male and female Sprague-Dawley rats at dose levels of 0, 500, 1500 and 4000 ppm (equivalent to an average of 14, 39 and 109 mg a.i./kg bw/day for males and 18, 51 and 137 mg a.i./kg bw/day in females, calculated from the mean achieved dose levels in the parental generation). Doses were administered before and throughout mating and gestation until the end of the lactation period in both F0 and F1 generations. At 4000 ppm, F0 and F1 parents showed significantly lower body weight gains and reduced food consumption. At 1500 or 500 ppm, no relevant changes were noted. Treatment with the test substance had no effect on the reproductive parameters in F0 and F1 parental rats at treatment levels up to 4000 ppm. No effect was observed on mating, fertility, gestation, fecundity or delivery at any concentration for either generation. No effect was recorded on litter parameters and on pre- and post-natal development of either generation at any concentration. Apart from cortical cell hypertrophy found in the adrenal glands of F0 females treated at 4000 ppm and lower spleen weight in F1 pups of the same group, no other treatment-related findings were seen upon microscopic examination of the concerned tissues. Consequently, under the study conditions, the NOAEL for parental toxicity was 1500 ppm (39-51 mg a.i./kg bw/day) for the male and female animals. The NOAEL for mating behaviour, fertility and gestation of each generation and for development, growth and survival of each progeny was 4000 ppm (109-137 mg a.i./kg bw/day) (Chevalier, 2008).   

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

30 Oct, 2003 - 21 May, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Deficiencies: Yes F0 animals: the day 11 samples taken for stability in closed bags and open feeders, were actually sampled on day 12 by error, males D26143 and D26154 given 1500 ppm were weighed again on day 17 due to aberrant values on day 15, for females D26364, pup No. 5 and D26393, pup No. 10 (given respectively 500 and 1500 ppm), macroscopic examination was performed although not scheduled in the study plan (data not presented in the study report), Male D26146 (given 1500 ppm): left testis and epididymis were mislaid before microscopic examination. F1 animals: according to the Study plan, the dosing period was 20 weeks. However, the study was finished in week 18, the dietary admixtures were analyzed in week 18 rather than week 20, as specified in the Study plan, because the study finished early, female D26509 given 4000 ppm and females D26439 and D26446 given 0 ppm were mated one extra day by error (the females had been positive for mating the previous day), during the mating periods (F0 and F1 animals), the food consumption was not recorded (typing error in the final study plan), organ weights with aberrant values have been excluded from mean calculation: thyroid glands of male D26210 (given 500 ppm), adrenal glands of male D26232 (given 1500 ppm) and pituitary gland of male D26234 (given 1500 ppm), male D26226 (given 500 ppm): dilated renal pelvis was observed for left kidney at macroscopic examination but no microscopic examination was performed, male D26262 (given 4000 ppm): adrenal glands were not found for the preparation of slides, female D26457 (given 500 ppm): pituitary gland was mislaid after weighing, female D26493, pup No. 4 (given 1500 ppm) found dead on day 2 post-partum was mislaid before macroscopic examination. These deviations were considered not to have compromised the validity or integrity of the study

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Rat
Strain: Sprague-Dawley, Crl CD® (SD) IGS BR
Source: Charles River Laboratories France, L’Arbresle, France.
Sex: 100 males and 100 females
Age/weight at study initiation: At the beginning of the treatment period, the animals were approximately 5 to 6 weeks old and had a mean body
weight of 137 g (range: 104 g to 168 g) for the males and 118 g (range: 96 g to 139 g) for the females.
Number of animals per group: 25 males and 25 females
Duration of mating: 2 weeks
Deviations from standard protocol: Female D26509 given 4000 ppm and females D26439 and D26446 given 0 ppm were mated one extra day
by error (the females had been positive for mating the previous day).
Control animal: Yes

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Administration/ Exposure: Oral
Duration of exposure before mating: 10 weeks
Duration of exposure in general P, F1, F2 males, females From beginning of the study until sacrifice of Parent, F1, F2-generation
Type: oral In food
Concentration Food consumption per day: 0, 500, 1500 and 4000 ppm ad libitum, for groups 1, 2, 3 and 4, respectively.
Vehicle: The test item was mixed in the powdered maintenance diet UAR A04C P2.5 (UAR, Villemoisson, Epinay-sur-Orge, France).
Controls: Vehicle

Details on mating procedure:

Males and females were paired for, at most, a 2-week period, until mating was obtained.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Doses were verified for test substance concentrations in regular intervals during the study.

Duration of treatment / exposure:

Exposure: Premating exposure period (males): 10 weeks
Premating exposure period (females): 10 weeks
Duration of test: F0 pre-mating 10 weeks, until F2 weaning. (Continuously)

Frequency of treatment:

Continuously

Details on study schedule:

F0 generation: 10 weeks before mating, during the mating period until sacrifice (weaning of pups) for the males, 10 weeks before mating, during the mating, pregnancy and lactation periods (until day 21 post-partum) for the females.

A group of 25 males and 25 females received untreated diet alone, under the same experimental conditions, and acted as a reference control group. 

F1 generation: At weaning of the F1 generation, on day 22 post-partum, three groups of 25 male and 25 female Sprague-Dawley rats received the same test item, under the same experimental conditions as above, during their growth, adulthood, mating, pregnancy and lactation, until weaning of the pups (F2 generation).

A group of 25 males and 25 females received untreated diet alone under the same experimental conditions and acted as a reference control group. 

Remarks:

Doses / Concentrations:
500, 1500, and 4000 ppm of test substance (40% active DDAC) (i.e., corresponding to 203, 608 and 1620 ppm of DDAC)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
14, 39 and 109 mg a.i./kg bw/day for males and 18, 51 and 137 mg a.i./kg bw/day in females
Basis:
nominal in diet

No. of animals per sex per dose:

25 per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

Refer to study design above

Positive control:

Not applicable

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, at most, a 2-week period, until mating was obtained.  The F0 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded. The F0 parent males and females were sacrificed after weaning of their progeny. During lactation, the pups from the F1 generation were observed daily for survival and clinical signs. Body weight was measured at designated intervals and 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 endpoints.

Oestrous cyclicity (parental animals):

Refer to study design

Sperm parameters (parental animals):

Refer to study design

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. Neurobehavioral tests were conducted at designated intervals to assess auditory and visual function. Spontaneous locomotor activity was also evaluated twice over a 60-min interval when the animals were 7 and 8 weeks old. When the animals were 12 weeks old, F1 males and F1 females were paired.
The F1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded. During lactation, the pups from 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 endpoints.

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 in 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 female killed at weaning.  Any pups which died or were killed prematurely during the lactation period were also submitted for 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. A detailed histopathological examination was performed on the ovaries and the testes.

Postmortem examinations (offspring):

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 in 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 female killed at weaning.  Any pups which died or were killed prematurely during the lactation period were also submitted for 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. A detailed histopathological examination was performed on the ovaries and the testes.

Statistics:

No data

Reproductive indices:

Please refer to study design for F0 animals

Offspring viability indices:

Please refer to study design for F1 animals

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

F0 generation
Test group 4 (4000 ppm): - no treatment-related deaths and no remarkable clinical signs,  - for the males, a lower body weight gain during the premating period (days 1-15), and food consumption was affected throughout the study,  - for the females, a lower body weight gain was noted during the first week of the premating period, during the gestation and the lactation, and food consumption was affected from week 2 of the study, but no effect was detected during the lactation, - there were no apparent effects on mating, fertility, fecundity or delivery,  - the pup body weight gain was significantly lower than controls from day 4 p.n.,  - no disturbances of the reflex development in the pups,  - none of the seminology parameters evaluated in F0 parents males were affected,  - distension of cecum with feces was seen at the autopsy in 13/25 F0 males, with no histopathological findings in this organ,  - higher adrenal weights (p<0.01) in F0 females correlated with enlargement of the gland and cortical cell hypertrophy seen at histopathology, - lower spleen weights in male and female pups sacrificed at weaning (p<0.01),  - no treatment-related findings were observed in male and female genital organs, and the accessory organs.          
Test group 3 (1500 ppm): - no treatment-related deaths, - no remarkable clinical signs, - neither body weight gain nor food consumption of parent animals were affected, - there were no apparent effects on mating, fertility, fecundity or delivery, - no effect on the pup body weight gain, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F0 parents males were affected, - no treatment-related differences in organ weights were recorded, - neither macroscopic nor microscopic findings were observed.
Test group 2 (500 ppm): - no treatment-related deaths, - no remarkable clinical signs, - neither body weight gain nor food consumption of parent animals were affected, - there were no apparent effects on mating, fertility, fecundity or delivery, - no effect on the pup body weight gain, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F0 parents males were affected, - no treatment-related differences in organ weights were recorded, - neither macroscopic nor microscopic findings were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

F1 generation
Test group 4 (4000 ppm): - no treatment-related deaths, - no remarkable clinical signs, - for the males, there was a lower body weight gain until day 43 of the premating period and food consumption was affected, mainly during the same period of the study, - for the females, a lower body weight gain was noted during the first 2 weeks of the premating period, from GD 7 (GD: gestation day) and during the lactation. The food consumption was affected during the premating period, during GD 0-14 and during days 7 14 p.p., - there were no apparent effects on mating, fertility, fecundity or the progress of delivery,  - the pup body weight gain was significantly lower than controls during the lactation, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F1 parents males were affected, - none of the organ weight differences recorded in F1 parents were directly treatment-related, - lower spleen weights were recorded in F1 pups, - no relevant findings were seen at necropsy of parents or pups, - neither macroscopic nor microscopic findings were observed.
Test group 3 (1500 ppm): - no treatment-related deaths, - no remarkable clinical signs, - neither body weight gain nor food consumption of parent animals were affected, - there were no apparent effects on mating, fertility, fecundity or the progress of delivery, - increase in the male ratio, - no effect on the pup body weight gain, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F1 parents males were affected. - no treatment-related differences in organ weights were recorded, - neither macroscopic nor microscopic findings were observed.
Test group 2 (500 ppm): - no treatment-related deaths, - no remarkable clinical signs, - neither body weight gain nor food consumption of parent animals were affected, - there were no apparent effects on mating, fertility, fecundity or the progress of delivery, - increase in the male ratio, - no effect on the pup body weight gain, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F1 parents males were affected. - no treatment-related differences in organ weights were recorded, - neither macroscopic nor microscopic findings were observed.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects on mating behaviour, fertility and gestation, of each generation and for development, growth and survival of each progeny

Key result

Reproductive effects observed:

no

Result: Not toxic to reproduction
concentrations of 500, 1500 or 4000 ppm test substance,
corresponding to 203, 608 or 1620 ppm of DDAC.

The estimation of the achieved dosages of test item
(expressed as mg of DDAC/kg/day) are summarized in the
following table:
Mean achieved dosages (in mg DDAC/kg/day)
Concentration (ppm)         500         1500         4000
F0 generation
Males
. premating (days 1-71)         17         49         137
. post-mating (days 85-120)     10         30         84
Females
. premating (days 1-71)         20         58         157
. gestation (GD 0-20)          15         45         52
. lactation (days 1-21 p.n.)    31         93         261
F1 generation
Males
. premating (days 1-64)         22         65         183
. post-mating (days 85-120)     10         31         94
Females
. premating (days 1-64)         23         69         198
. gestation (GD 0-20)          17         48         57
. lactation (days 1-21 p.n.)    36         93         263

Conclusions:

Based on the results of the read across study, the NOAEL for parental toxicity was 1500 ppm (39-51 mg a.i./kg bw/day) for the male and female animals. The NOAEL for mating behaviour, fertility and gestation of each generation and for development, growth and survival of each progeny was 4000 ppm (109-137 mg a.i./kg bw/day).

Executive summary:

A two-generation study was conducted to determine the toxicity to reproduction of the read across substance, DDAC (40% active in water) according to OECD Guideline 416, in compliance with GLP. In this study, the substance was administered in the diet to male and female Sprague-Dawley rats at dose levels of 0, 500, 1500 and 4000 ppm (equivalent to an average of 14, 39 and 109 mg a.i./kg bw/day for males and 18, 51 and 137 mg a.i./kg bw/day in females, calculated from the mean achieved dose levels in the parental generation). Doses were administered before and throughout mating and gestation until the end of the lactation period in both F0 and F1 generations. At 4000 ppm, F0 and F1 parents showed significantly lower body weight gains and reduced food consumption. At 1500 or 500 ppm, no relevant changes were noted. Treatment with the read across substance had no effect on the reproductive parameters in F0 and F1 parental rats at treatment levels up to 4000 ppm. No effect was observed on mating, fertility, gestation, fecundity or delivery at any concentration for either generation. No effect was recorded on litter parameters and on pre- and post-natal development of either generation at any concentration. Apart from cortical cell hypertrophy found in the adrenal glands of F0 females treated at 4000 ppm and lower spleen weight in F1 pups of the same group, no other treatment-related findings were seen upon microscopic examination of the concerned tissues. Consequently, under the study conditions, the NOAEL for parental toxicity was 1500 ppm (39-51 mg a.i./kg bw/day) for the male and female animals. The NOAEL for mating behaviour, fertility and gestation of each generation and for development, growth and survival of each progeny was 4000 ppm (109-137 mg a.i./kg bw/day) (Chevalier, 2008). Based on the results of the read across study, similar absence of reproductive toxicity is expected for the test substance. 

Reference 3

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Not reported

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
No data

Details on mating procedure:

No data

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

Exposure period: Continuous
Premating exposure period (males): 10 wk F1A, and approx. 19 wk F1B
Premating exposure period (females): 10 wk F1A, and approx. 19 wk F1B
Duration of test: Weaning second generation

Details on study schedule:

Exposures continued through mating, gestation, parturition and lactation
- At least 10 d after weaning the F1A litters, FO parents were mated in different male-female pairings, within dose groups, to produce the F1B generation.

Remarks:

Doses / Concentrations:
0, 300, 750 and 1500 ppm
Basis: nominal in diet

No. of animals per sex per dose:

28/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

No data

Positive control:

Not used in the study

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

No data

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- High dose and control animals

Postmortem examinations (offspring):

No data

Statistics:

Not reported

Reproductive indices:

No data

Offspring viability indices:

No data

Clinical signs:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight reduction

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Body weight reduction

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Body weight reduction, weight gain depression and decreased food consumption at 1500 ppm

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No adverse effects

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Clinical signs:

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

BODY WEIGHT (OFFSPRING): Reduced pup body weights at 1500 ppm

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Reproductive effects observed:

no

The details of the study were reproduced from the original report of Bradley NTL (1991).

Conclusions:

Under the study conditions, the NOEL for both adults and offspring was 750 ppm, indicating no increased risk to offspring in the absence of indications of adult toxicity.

Executive summary:

A two-generation study was conducted to determine the toxicity to reproduction of the test substance, DDAC (purity not specified) according to a method similar to OECD Guideline 416. In this study, the substance was administered in the diet to 28 Sprague-Dawley rats/sex/dose at dose levels of 0, 300, 750, or 1500 ppm (equivalent to 112.6 mg/kg bw/day). Dose levels in terms of mg/kg bw/day decreased throughout the study as animal body weight increased and varied within and between dose groups and generations, so are not reported here. Animals were exposed to test material for 10 wk prior to mating, and each of the two generations produced two litters. The rats in F0 generation, were randomly paired within dose groups and mated over a 3 week period to produce the F1A generation. Exposures continued through mating, gestation, parturition, and lactation. At least 10 day after weaning the F1A litters, F0 parents were mated in different male-female pairings, within dose groups, to produce the F1B generation. The test substance was then exposed from mating to lactation. After the F1B animals were weaned, F0 parents were necropsied and high dose and control animals were examined for histopathologic lesions. Selected F1 parents were exposed to the same concentrations of test substance as their parents for at least 10 week, and were then paired as described above to produce F2A and F2B generations. Mating, gestation, lactation, and necropsy of the F1 parents and selected F2A and F2B pups were performed as outlined above, except that no F2 animals were selected as parents. Continuous exposure to test substance in the diet for two generations resulted in no adverse reproductive effects. Parental toxicity was observed at 1500 ppm (112.6 mg/kg bw/day), limited to body weight reduction, weight gain depression, and decreased food consumption. Postnatal toxicity at 1500 ppm was indicated by reduced pup body weights. Under the study conditions, the NOEL for both adults and offspring was 750 ppm, indicating no increased risk to offspring in the absence of indications of adult toxicity (Henderson, 1992).

Reference 4

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Not reported

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
No data

Details on mating procedure:

No data

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

Exposure period: Continuous
Premating exposure period (males): 10 wk F1A, and approx. 19 wk F1B
Premating exposure period (females): 10 wk F1A, and approx. 19 wk F1B
Duration of test: Weaning second generation

Details on study schedule:

Exposures continued through mating, gestation, parturition and lactation
- At least 10 d after weaning the F1A litters, FO parents were mated in different male-female pairings, within dose groups, to produce the F1B generation.

Remarks:

Doses / Concentrations:
0, 300, 750 and 1500 ppm
Basis: nominal in diet

No. of animals per sex per dose:

28/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

No data

Positive control:

Not used in the study

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

No data

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- High dose and control animals

Postmortem examinations (offspring):

No data

Statistics:

Not reported

Reproductive indices:

No data

Offspring viability indices:

No data

Clinical signs:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight reduction

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Body weight reduction

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Body weight reduction, weight gain depression and decreased food consumption at 1500 ppm

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No adverse effects

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Clinical signs:

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

BODY WEIGHT (OFFSPRING): Reduced pup body weights at 1500 ppm

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Reproductive effects observed:

no

The details of the study were reproduced from the original report of Bradley NTL (1991).

Conclusions:

Based on the results of read across study, the NOEL for both adults and offspring is considered 750 ppm, indicating no increased risk to offspring in the absence of indications of adult toxicity.

Executive summary:

A two-generation study was conducted to determine the toxicity to reproduction of the read across substance, DDAC (purity not specified) according to a method similar to OECD Guideline 416. In this study, the substance was administered in the diet to 28 Sprague-Dawley rats/sex/dose at dose levels of 0, 300, 750, or 1500 ppm (equivalent to 112.6 mg/kg bw/day). Dose levels in terms of mg/kg bw/day decreased throughout the study as animal body weight increased and varied within and between dose groups and generations, so are not reported here. Animals were exposed to test material for 10 wk prior to mating, and each of the two generations produced two litters. The rats in F0 generation, were randomly paired within dose groups and mated over a 3 week period to produce the F1A generation. Exposures continued through mating, gestation, parturition, and lactation. At least 10 day after weaning the F1A litters, F0 parents were mated in different male-female pairings, within dose groups, to produce the F1B generation. The read across substance was then exposed from mating to lactation. After the F1B animals were weaned, F0 parents were necropsied and high dose and control animals were examined for histopathologic lesions. Selected F1 parents were exposed to the same concentrations of read across substance as their parents for at least 10 week, and were then paired as described above to produce F2A and F2B generations. Mating, gestation, lactation, and necropsy of the F1 parents and selected F2A and F2B pups were performed as outlined above, except that no F2 animals were selected as parents. Continuous exposure to read across substance in the diet for two generations resulted in no adverse reproductive effects. Parental toxicity was observed at 1500 ppm (112.6 mg/kg bw/day), limited to body weight reduction, weight gain depression, and decreased food consumption. Postnatal toxicity at 1500 ppm was indicated by reduced pup body weights. Under the study conditions, the NOEL for both adults and offspring was 750 ppm, indicating no increased risk to offspring in the absence of indications of adult toxicity (Henderson, 1992). Based on the results of the read across study, similar absence of reproductive toxicity is expected for the test substance. 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Well documented study, meets generally accepted scientific principles, acceptable for assessment. No true systemic effects; systemic NOAELs not used futher for risk assessment.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No reproductive toxicity study could be located on C12-18 DAQ. Therefore, read across studies available with the structurally similar substance DDAC is presented. Both the test and read across substances are di-alkyl dimethyl ammonium chloride compounds. DDAC is structurally the same but only differs in a slightly lower average alkyl chain length. Slightly shorter alkyl chains do not change possible chemical reactivity, but DDAC with a smaller molecule is potentially more bioavailable, and thus is more likely to show effects, therefore representing a worst case. 

 

Study 1: A two-generation study was conducted to determine the toxicity to reproduction of the read across substance, DDAC (40% active in water) according to OECD Guideline 416, in compliance with GLP. In this study, the substance was administered in the diet to male and female Sprague-Dawley rats at dose levels of 0, 500, 1500 and 4000 ppm (equivalent to an average of 14, 39 and 109 mg a.i./kg bw/day for males and 18, 51 and 137 mg a.i./kg bw/day in females, calculated from the mean achieved dose levels in the parental generation). Doses were administered before and throughout mating and gestation until the end of the lactation period in both F0 and F1 generations. At 4000 ppm, F0 and F1 parents showed significantly lower body weight gains and reduced food consumption. At 1500 or 500 ppm, no relevant changes were noted. Treatment with the read across substance had no effect on the reproductive parameters in F0 and F1 parental rats at treatment levels up to 4000 ppm. No effect was observed on mating, fertility, gestation, fecundity or delivery at any concentration for either generation. No effect was recorded on litter parameters and on pre- and post-natal development of either generation at any concentration. Apart from cortical cell hypertrophy found in the adrenal glands of F0 females treated at 4000 ppm and lower spleen weight in F1 pups of the same group, no other treatment-related findings were seen upon microscopic examination of the concerned tissues. Consequently, under the study conditions, the NOAEL for parental toxicity was 1500 ppm (39-51 mg a.i./kg bw/day) for the male and female animals. The NOAEL for mating behaviour, fertility and gestation of each generation and for development, growth and survival of each progeny was 4000 ppm (109-137 mg a.i./kg bw/day) (Chevalier, 2008).  

Study 2: A two-generation study was conducted to determine the toxicity to reproduction of the read across substance, DDAC (purity not specified) according to a method similar to OECD Guideline 416. In this study, the substance was administered in the diet to 28 Sprague-Dawley rats/sex/dose at dose levels of 0, 300, 750, or 1500 ppm (equivalent to 112.6 mg/kg bw/day). Dose levels in terms of mg/kg bw/day decreased throughout the study as animal body weight increased and varied within and between dose groups and generations, so are not reported here. Animals were exposed to test material for 10 wk prior to mating, and each of the two generations produced two litters. The rats in F0 generation, were randomly paired within dose groups and mated over a 3 week period to produce the F1A generation. Exposures continued through mating, gestation, parturition, and lactation. At least 10 day after weaning the F1A litters, F0 parents were mated in different male-female pairings, within dose groups, to produce the F1B generation. The read across substance was then exposed from mating to lactation. After the F1B animals were weaned, F0 parents were necropsied and high dose and control animals were examined for histopathologic lesions. Selected F1 parents were exposed to the same concentrations of read across substance as their parents for at least 10 week, and were then paired as described above to produce F2A and F2B generations. Mating, gestation, lactation, and necropsy of the F1 parents and selected F2A and F2B pups were performed as outlined above, except that no F2 animals were selected as parents. Continuous exposure to read across substance in the diet for two generations resulted in no adverse reproductive effects. Parental toxicity was observed at 1500 ppm (112.6 mg/kg bw/day), limited to body weight reduction, weight gain depression, and decreased food consumption. Postnatal toxicity at 1500 ppm was indicated by reduced pup body weights. Under the study conditions, the NOEL for both adults and offspring was 750 ppm, indicating no increased risk to offspring in the absence of indications of adult toxicity (Henderson, 1992).

Further, the DDAC assessment report for Product Type 8 conducted under Directive 98/8/EC (evaluating Competent Authority: Italy, June 2015, attached in Section 13 of the IUCLID dataset) reported one additional 2-generation study in rats. In this study, based on reduced body weight and food consumption in F0 and F1 animals, the maternal NOAEL was established at 750 ppm (≥31 mg/kg bw/day). In the offsprings, except for reduced body weight there were no other adverse effects leading to establishment of NOAEL at 750 ppm (≥31 mg/kg bw/day). There were also no treatment related effects on any reproductive parameters were observed at any dose level. Further, the biocides assessment report, cited slightly different doses in mg/kg bw/day for the EQC (Chevalier 2008) study. The parental, fertility and offspring NOAELs were all established at 608 ppm (>30 mg/kg bw/day). The RMS further stated that:“Available studies do not indicate any specific potential for reproductive toxicity. Observed effects concern solely general toxicity”. The overall NOAEL (parental effects) was concluded to be 30 mg/kgbw/day, but it was not considered relevant for systemic toxicity.

Therefore, in line with the biocides assessment report, the NOAELs from the 2-generation reproductive toxicity studies in rats have not been considered further for systemic risk assessment. 

Effects on developmental toxicity

Description of key information

The available oral and dermal pre-natal development toxicity study with the read across substances in rats and/or rabbits, indicate no concern for development toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Elevage Scientifique des Dombes, Châtillon-sur-Chalaronne, France)
- Age at study initiation: 18-20 wk
- Weight at study initiation: mean body weight of 3494 g (range: 3105 g to 3940 g).
- Housing: individually housed in stainless steel cages, in a barriered rabbit unit, under specific pathogen free (SPF) standard laboratory

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3°C
- Humidity: 50 ± 20%
- Air changes: 8 to 10 cycles/h of filtered, non-recycled air
- Photoperiod: 8h dark/16h light (5:00 - 21:00)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Concentration in vehicle: 0, 1.33, 4, 10.66 mg/mL. The test item dosage forms were prepared daily and stored at room temperature prior to use.
Total volume applied: 3 mL/kg/d

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of DDAC in dosage form: The concentration of samples taken from each control and test item dosage form prepared for use on the first day of treatment and on the last day of treatment was determined. Values were found to be in agreement with nominal concentration ± 10%.

Details on mating procedure:

Animals were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as day 0 post-coitum (p.c.) or day 0 of gestation (GD 0).

Duration of treatment / exposure:

Day 6 to 28 post coitum

Frequency of treatment:

Daily

Duration of test:

Upto 28 d of gestation

Remarks:

Doses / Concentrations:
0, 4, 12 and 32 mg DDAC/kg/d
Basis:
nominal conc.

No. of animals per sex per dose:

22 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a preliminary study of effects on embryo-fetal development in rabbits (CIT/Study No. 26153 RSL), in which the test item, DDAC, was administered to pregnant female rabbits, from Day 6 to Day 28 of pregnancy at the dose-levels of 4, 16 or 32 mg DDAC/kg/d.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day 2, 4, 5, 6, 9, 12, 15, 18, 22, 26 and 29 post-coitum

FOOD CONSUMPTION: Yes
- Recorded for the following intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-18, 18-22, 22-26 and 26-29 post-coitum.
Any obvious spillage of food was documented.

POST-MORTEM EXAMINATIONS: Yes

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes; all per litter
- Soft tissue examinations: Yes; all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
- Examination of fetuses: Carried out only in the first 16 litters (females with at least one live fetus). The other litters will be discarded without further investigations.

Statistics:

Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.

Indices:

Assessment of data
Data are expressed as group mean values ± standard deviation (maternal body weight and food consumption, fetal body weight, number of corpora lutea, implantations, fetuses and resorptions) or as proportions (pre-implantation loss, post-implantation loss and fetal findings). Whenever necessary, the experimental unit of comparison was the litter. Data on non-pregnant females were not included in the group mean calculations.

The following calculations were made:
Net body weight (presented as carcass weight): body weight on day 29 minus gravid uterine weight.
Net body weight change: (body weight on day 29 minus body weight on day 6) minus gravid uterine weight.

Pre-implantation loss: Number of corpora lutea - Number of implantation sites x 100 / Number of corpora lutea
Post-implantation loss: Number of implantation sites - Number of live fetuses x 100 / Number of implantation sites
Fetal or litter incidence: Total number of fetuses or litters with a particular finding x 100 / Total number of fetuses or litters examined
Mean proportion of affected fetuses: Sum of proportion of fetuses affected in each litter x 100 / Total number of litters examined

Historical control data:

No data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

When given 12 mg DDAC/kg/day, colored urine and loud breathing was observed in one female from GD 22. These signs were attributed to treatment with the test item. At 32 mg DDAC/kg/day, the number of animals showing clinical signs was greater than in the control group, and most of these clinical signs (colored urine, soft feces, soiled urogenital area) were recorded at the end of the gestation period, along with an absence of feces observed for three animals. Presence of blood in the bedding was noted in two females on GD 16 or 22 and one female was emaciated (E30669, GD 29). No other clinical signs were considered to be treatment-related, absence of feces is commonly recorded at the end of pregnancy and was therefore not considered to be treatment-related at 4 mg DDAC/kg/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

32 mg DDAC/kg/day group
Female E30668 was found dead after treatment on GD 25. Abdominal breathing from GD 24 and coldness to the touch on GD 25 were noted. During GD 18-22, food consumption was reduced and the animal lost 210 g. Dilated stomach with liquid and findings in the lungs (brownish foci, reddish areas and firm consistency) were seen at necropsy. Female E30673 was prematurely killed on GD 22 following signs of poor health status (hypotonia and coldness to the touch on GD 22). Liquid feces were seen from GD 20, soiled urogenital area from GD 21 and blood in bedding on GD 22. This animal lost weight from GD 6 (GD 6-22: -485 g), but the body weight loss was more pronounced from GD 18 (GD 18-22:-235 g) and it did not eat from GD 15. At necropsy, gall bladder was dilated and cecum was distended with gas. Female E30687 was found dead on GD 22, one fetus was found in the bedding on that day and blood in the bedding was noted from GD 21. Liquid feces with soiled urogenital area were seen from GD 20. During GD 15-18, food consumption was reduced and the animal lost 170 g. Numerous findings were detected at necropsy in the stomach (brownish foci, deposit in the mucosa) and in the intestines (cecum distended with gas and liquid contents). The gall bladder was dilated.

12 mg DDAC/kg/day group
Female E30652 aborted on GD 28. Absence of feces were noted from GD 22. During GD 15-26 this animal lost 380 g and ate only 26 g/day. At autopsy, the intestines were dilated (brown liquid content in cecum) and the gall bladder was reduced in size.

4 mg DDAC/kg/day group
Animal E30629 was found dead on GD 10. Gavage of this animal was difficult on GD 9 (traces of blood were in evidence on the gavage tube). On GD 10, the animal presented loud and abdominal breathing and oral administration of the dosage form was not possible. During GD 6-9, this animal lost 135 g and ate only 48 g. The autopsy revealed brownish areas in the lung lobes. Taking into account that this death occurred after gavage difficulties, a direct relationship to DDAC was improbable, and this death was considered incidental. Female E30633 was found dead on GD 26 without ante-mortem clinical signs. Food consumption and body weight change were normal on the days preceeding death. Necropsy of the lungs showed many brownish foci, several reddish areas and a firm consistency of the organ. In view of these last findings, this death could be consecutive to accidental aspiration of the test item into the lungs. Therefore a direct relationship to repeated administration of DDAC could be ruled out.

Control group
Female E30618 aborted on GD 23 (8 placentas in the bedding). Abnormal breathing was recorded from GD 18. Absence of feces and blood in the bedding were observed from GD 21. From GD 15, a marked reduction in food consumption (GD 15-18: 70 g/day, GD 18-22: 8 g/day) and a body weight loss (GD 15-22: -660 g) were recorded. No findings were seen at the autopsy. This abortion was considered to be spontaneous.

In absence of a dose-related increase of death/abortion at 4 and 12 mg DDAC/kg/day and with the abortion of one female in the control group, the mortality at 4 and 12 mg DDAC/kg/day were considered to be most probably incidental and not related to treatment with the test item. At 32 mg DDAC mg/kg/day, death/abortion in 3 females were ascribed to the treatment with the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 32 mg/kg/day, a body weight loss was noted from GD 6 to 9 and concerned ten females in comparison with two females of the control group. Therefore, the body weight gains during all intervals were lower than in the control group, showing statistical significance from GD 6 to 12, GD 6 to 15 and GD 6 to 26. At 32 mg/kg/day, the body weight gain during the whole dosing period was statistically lower (-56%, p<0.01) than in the control group. At 12 mg/kg/day, the body weight gain was slightly reduced during many intervals of the dosing period, reaching statistical differences from GD 6 to 15 and GD 6 to 26 and corresponds to -29% from GD 6 to 29 (whole dosing period) when compared to controls. At 4 mg/kg/day, no marked effects were noted in the body weight gains. The net body weight change (reflecting the maternal body weight change independently of the uterus weight) was lower in the treated groups than in the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 32 mg/kg/day, the mean food consumption was lower than the treated group values during the whole dosing period, differences being more marked at the beginning and at the end of the dosing period (GD 6-9, -22%, p<0.001; GD 9-12, -18%, p<0.001; GD 26-29, -37%, not statistically significant when compared to control values). At 4 and 12 mg/kg/day, very slight reductions were noted at different intervals, none of them were statistically significant when compared to control values.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The uterus weight of treated females at the low dose-level of DDAC was similar to that of the control group, while the weight of the gravid uterus of treated females at 12 and 32 mg DDAC/kg/day was slightly lower. These differences were correlated with lower numbers of fetuses.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy findings in the intestines (all treated groups), stomach and gall bladder (high-dose group only) were considered to be related to the treatment with DDAC. No other treatment-related findings were observed. There were no findings at the gross examination of placentae in any females.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

When compared with the control value, the number of dead fetuses was higher (and statistically significant when expressed as a total number) at 32 mg DDAC/kg/day. This difference led to a lower number of live fetuses, and a high mean post-implantation loss. All these mean values for this group were outside CIT background data. It is worth noting that the number of autolyzed fetuses was higher than in the control group.
The high number of autolyzed fetuses recorded in group 4 (32 mg DDAC/kg/day) was due to three litters (E30667, E30669 and E30670) with 5, 13 and 6 autolyzed fetuses. Although the number of dead fetuses recorded in the control group was higher than the CIT background data (mean per female= 0.2), all the differences noted in the litter data described above for the group treated at 32 mg DDAC/kg/day was attributed to treatment with the test item. The modification of these parameters in the low dose group was not considered to be treatment-related in the absence of obvious dose-relationship. The number of live fetuses in the intermediate dose-group and the control group were similar. The percentage of male fetuses and the fetal body weights were unaffected by treatment.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
32 mg DDAC/kg/d:
Evident signs of maternal toxicity (3 mortality), abortion, clinical signs at the end of the gestation; reduction in food consumption and body weight loss) were noted. Necropsy findings concerned the intestines, the stomach and the gall bladder.
Clinical signs were observed in 11/19 animals and consisted of colored urine (5/19), soft feces (4/19), absence feces (3/19), blood in bedding (2/19), and one incidence of loud breathing. One animal showed an emaciated appearance at the end of the study. Autopsy of the surviving animals revealed effects on intestines (8/19: distended with gas, dilated, brownish content), stomach (2/19: dilated, thickened wall, brown/whitish area/deposit), gall bladder (1/19: dilated) and lungs (1/19: brownish foci and reddish areas).

12 mg DDAC/kg/d:
One animal aborted on GD28 after period with absence of feces, weight loss, and autopsy showed dilated intestines and gall bladder reduced in size. One animal was not pregnant. Clinical signs in the remaining surviving animals consisted of absence of feces at end of pregnancy (2/10), loud breathing (1/20) and reddish colored urine from GD22 (1/20). Observations at autopsy: brownish foci in lungs (1/20), accentuated lobular pattern liver (2/10), thickened wall uterus (2/10), and brownish content intestines with liquid in cecum (2/10).

4 mg DDAC/kg/d:
Two animals found dead (GD 10 and 26) both displaying brownish areas/foci in lungs indicating possible aspiration of test item. Clinical signs in the remaining pregnant animals comprised of absence of feces observed at GD29 or from GD28 (3/20). Autopsy showed brownish foci in lungs (1/20), effects on intestines (2/20), and a pale liver (1/20).

Control:
One animal aborted on GD23 after a period of reduced food consumption, abnormal breathing, absence of feces, and blood in bedding. No autopsy findings. Of the remaining pregnant animals at term observations included blood in bedding (2/21), cutaneous lesion (2/20) and immobilized hind limb (1/20). Autopsy showed brownish foci in lungs in 3/20. (Net) body weight changes are difficult to interpret due to the big inter-individual variation. There are also large intra-individual differences between periods of the gestation period. Overall, there seems to be a dose related effect, tending to lower body weights at higher dose levels which corresponding to lower food consumption.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 4 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: effects secondary to local effects

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The fetal body weights were unaffected by treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The percentage of male fetuses were unaffected by treatment.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of fetuses showing external malformations was 1, 0, 2 and 0 distributed in 1, 0, 2 and 0 litters at 0, 4, 12 or 32 mg DDAC/kg/day. This distribution was not considered to be treatment-related. Variations were also distributed in different groups including control group and with no significant dose-related increase.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No dose- or treatment-related increases were noted for any skeletal malformations. At 32 mg DDAC/kg/day, the number of litters with fetuses with incomplete ossification of the sixth sternebrae was higher than control (p<0.05 when expressed as litter incidence). Taking into account that this variation is commonly observed in fetuses of this strain of rabbit, and that incidences were within the CIT background data range, it was not considered to be a consequence of treatment with DDAC. No treatment-related changes were observed at cartilage examination in any of the litters examined. Therefore, it could be considered that the general ossification of fetuses was not affected by treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The only malformations detected at visceral examination were considered to be spontaneous in origin as affecting one fetus in the 0 and 12 mg DDAC/kg/day. The presence of whitish colored fluid in the abdomen (variation) of 5 fetuses from the same litter (5/6 fetuses doe E30667) treated at 32 mg DDAC/kg/day was considered to be most probably related to the test-treatment (statistically significant), as this finding was not observed in any of the fetuses from control group, and both the fetal and litter incidence were outside CIT background data.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 12 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: based on decreased number of live foetuses, increased post-implantation loss and a decreased foetal body weight at 32 mg/kg bw/day

Remarks on result:

other: prenatal effects only seen as unspecific consequence of maternal distress

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

32 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

yes

None

Conclusions:

Under the study conditions, the NOAEL for maternal toxicity and embryotoxic/teratogenic effects of the test substance were determined to be 4 and 12 mg a.i./kg bw/day respectively.

Executive summary:

A study was conducted to evaluate the prenatal developmental toxicity of the test substance, DDAC (40% active in water) in rabbits by oral gavage route, according to OECD guideline 414 and EPA OPPTS 870.3700, in compliance with GLP. The test substance at concentrations of 0, 4, 12 and 32 mg a.i./kg bw/day was administered by gavage, to groups of 20 female New Zealand White rabbits from Day 6 to 28 post coitum. All females were submitted to a macroscopic post-mortem examination of the principal thoracic and abdominal organs. A gross evaluation of placentas was also undertaken. Fetuses were examined for skeleton and soft tissue malformations. When given 12 mg a.i./kg bw/day, coloured urine and loud breathing was observed in one female from GD 22 each. Both animals however, showed no findings at autopsy. At 32 mg a.i./kg bw/day, the number of animals showing clinical signs was greater than in the control group, and most of these clinical signs (coloured urine, soft faeces, soiled urogenital area) were recorded at the end of the gestation period, along with an absence of faeces observed for three animals. Presence of blood in the bedding was noted in two females on GD 16 or 22 and one female showed an emaciated appearance on GD 29. There was a big variability in litter data. The low dose group showed higher number of dead fetuses, post implantation loss and lower fetal weight compared to control. On the other hand, the number of live fetuses was even higher than of control. At the mid-dose, observations were comparable to control again. The high-dose group of 32 mg a.i./kg bw/day resulted to an increased post-implantation loss, decrease in live fetuses with a decreased fetal body weight, and high number of dead fetuses. Upon examination of the soft tissue, presence of whitish fluid in the abdomens of 5/6 fetuses (one litter) was observed. No relevant malformations or variations were observed during the fetal evaluation. At lower concentrations, litter parameters were not affected by the test treatment and no evidence of treatment-related findings were noted at skeletal examinations of the fetuses. Overall, there seems to be a dose related effect, tending to lower body weights at higher dose levels which corresponding to lower food consumption related to palatability of the compound in the diet. Under the study conditions, the NOAEL for maternal toxicity and embryotoxic/teratogenic effects of the test substance were determined to be 4 and 12 mg a.i./kg bw/day respectively (Chevalier, 2005). 

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Elevage Scientifique des Dombes, Châtillon-sur-Chalaronne, France)
- Age at study initiation: 18-20 wk
- Weight at study initiation: mean body weight of 3494 g (range: 3105 g to 3940 g).
- Housing: individually housed in stainless steel cages, in a barriered rabbit unit, under specific pathogen free (SPF) standard laboratory

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3°C
- Humidity: 50 ± 20%
- Air changes: 8 to 10 cycles/h of filtered, non-recycled air
- Photoperiod: 8h dark/16h light (5:00 - 21:00)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Concentration in vehicle: 0, 1.33, 4, 10.66 mg/mL. The test item dosage forms were prepared daily and stored at room temperature prior to use.
Total volume applied: 3 mL/kg/d

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of DDAC in dosage form: The concentration of samples taken from each control and test item dosage form prepared for use on the first day of treatment and on the last day of treatment was determined. Values were found to be in agreement with nominal concentration ± 10%.

Details on mating procedure:

Animals were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as day 0 post-coitum (p.c.) or day 0 of gestation (GD 0).

Duration of treatment / exposure:

Day 6 to 28 post coitum

Frequency of treatment:

Daily

Duration of test:

Upto 28 d of gestation

Remarks:

Doses / Concentrations:
0, 4, 12 and 32 mg DDAC/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

22 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a preliminary study of effects on embryo-fetal development in rabbits (CIT/Study No. 26153 RSL), in which the test item, DDAC, was administered to pregnant female rabbits, from Day 6 to Day 28 of pregnancy at the dose-levels of 4, 16 or 32 mg DDAC/kg/d.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day 2, 4, 5, 6, 9, 12, 15, 18, 22, 26 and 29 post-coitum

FOOD CONSUMPTION: Yes
- Recorded for the following intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-18, 18-22, 22-26 and 26-29 post-coitum.
Any obvious spillage of food was documented.

POST-MORTEM EXAMINATIONS: Yes

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes; all per litter
- Soft tissue examinations: Yes; all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
- Examination of fetuses: Carried out only in the first 16 litters (females with at least one live fetus). The other litters will be discarded without further investigations.

Statistics:

Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.

Indices:

Assessment of data
Data are expressed as group mean values ± standard deviation (maternal body weight and food consumption, fetal body weight, number of corpora lutea, implantations, fetuses and resorptions) or as proportions (pre-implantation loss, post-implantation loss and fetal findings). Whenever necessary, the experimental unit of comparison was the litter. Data on non-pregnant females were not included in the group mean calculations.

The following calculations were made:
Net body weight (presented as carcass weight): body weight on day 29 minus gravid uterine weight.
Net body weight change: (body weight on day 29 minus body weight on day 6) minus gravid uterine weight.

Pre-implantation loss: Number of corpora lutea - Number of implantation sites x 100 / Number of corpora lutea
Post-implantation loss: Number of implantation sites - Number of live fetuses x 100 / Number of implantation sites
Fetal or litter incidence: Total number of fetuses or litters with a particular finding x 100 / Total number of fetuses or litters examined
Mean proportion of affected fetuses: Sum of proportion of fetuses affected in each litter x 100 / Total number of litters examined

Historical control data:

No data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

When given 12 mg DDAC/kg/day, colored urine and loud breathing was observed in one female from GD 22. These signs were attributed to treatment with the test item. At 32 mg DDAC/kg/day, the number of animals showing clinical signs was greater than in the control group, and most of these clinical signs (colored urine, soft feces, soiled urogenital area) were recorded at the end of the gestation period, along with an absence of feces observed for three animals. Presence of blood in the bedding was noted in two females on GD 16 or 22 and one female was emaciated (E30669, GD 29). No other clinical signs were considered to be treatment-related, absence of feces is commonly recorded at the end of pregnancy and was therefore not considered to be treatment-related at 4 mg DDAC/kg/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

32 mg DDAC/kg/day group
Female E30668 was found dead after treatment on GD 25. Abdominal breathing from GD 24 and coldness to the touch on GD 25 were noted. During GD 18-22, food consumption was reduced and the animal lost 210 g. Dilated stomach with liquid and findings in the lungs (brownish foci, reddish areas and firm consistency) were seen at necropsy. Female E30673 was prematurely killed on GD 22 following signs of poor health status (hypotonia and coldness to the touch on GD 22). Liquid feces were seen from GD 20, soiled urogenital area from GD 21 and blood in bedding on GD 22. This animal lost weight from GD 6 (GD 6-22: -485 g), but the body weight loss was more pronounced from GD 18 (GD 18-22:-235 g) and it did not eat from GD 15. At necropsy, gall bladder was dilated and cecum was distended with gas. Female E30687 was found dead on GD 22, one fetus was found in the bedding on that day and blood in the bedding was noted from GD 21. Liquid feces with soiled urogenital area were seen from GD 20. During GD 15-18, food consumption was reduced and the animal lost 170 g. Numerous findings were detected at necropsy in the stomach (brownish foci, deposit in the mucosa) and in the intestines (cecum distended with gas and liquid contents). The gall bladder was dilated.

12 mg DDAC/kg/day group
Female E30652 aborted on GD 28. Absence of feces were noted from GD 22. During GD 15-26 this animal lost 380 g and ate only 26 g/day. At autopsy, the intestines were dilated (brown liquid content in cecum) and the gall bladder was reduced in size.

4 mg DDAC/kg/day group
Animal E30629 was found dead on GD 10. Gavage of this animal was difficult on GD 9 (traces of blood were in evidence on the gavage tube). On GD 10, the animal presented loud and abdominal breathing and oral administration of the dosage form was not possible. During GD 6-9, this animal lost 135 g and ate only 48 g. The autopsy revealed brownish areas in the lung lobes. Taking into account that this death occurred after gavage difficulties, a direct relationship to DDAC was improbable, and this death was considered incidental. Female E30633 was found dead on GD 26 without ante-mortem clinical signs. Food consumption and body weight change were normal on the days preceeding death. Necropsy of the lungs showed many brownish foci, several reddish areas and a firm consistency of the organ. In view of these last findings, this death could be consecutive to accidental aspiration of the test item into the lungs. Therefore a direct relationship to repeated administration of DDAC could be ruled out.

Control group
Female E30618 aborted on GD 23 (8 placentas in the bedding). Abnormal breathing was recorded from GD 18. Absence of feces and blood in the bedding were observed from GD 21. From GD 15, a marked reduction in food consumption (GD 15-18: 70 g/day, GD 18-22: 8 g/day) and a body weight loss (GD 15-22: -660 g) were recorded. No findings were seen at the autopsy. This abortion was considered to be spontaneous.

In absence of a dose-related increase of death/abortion at 4 and 12 mg DDAC/kg/day and with the abortion of one female in the control group, the mortality at 4 and 12 mg DDAC/kg/day were considered to be most probably incidental and not related to treatment with the test item. At 32 mg DDAC mg/kg/day, death/abortion in 3 females were ascribed to the treatment with the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 32 mg/kg/day, a body weight loss was noted from GD 6 to 9 and concerned ten females in comparison with two females of the control group. Therefore, the body weight gains during all intervals were lower than in the control group, showing statistical significance from GD 6 to 12, GD 6 to 15 and GD 6 to 26. At 32 mg/kg/day, the body weight gain during the whole dosing period was statistically lower (-56%, p<0.01) than in the control group. At 12 mg/kg/day, the body weight gain was slightly reduced during many intervals of the dosing period, reaching statistical differences from GD 6 to 15 and GD 6 to 26 and corresponds to -29% from GD 6 to 29 (whole dosing period) when compared to controls. At 4 mg/kg/day, no marked effects were noted in the body weight gains. The net body weight change (reflecting the maternal body weight change independently of the uterus weight) was lower in the treated groups than in the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 32 mg/kg/day, the mean food consumption was lower than the treated group values during the whole dosing period, differences being more marked at the beginning and at the end of the dosing period (GD 6-9, -22%, p<0.001; GD 9-12, -18%, p<0.001; GD 26-29, -37%, not statistically significant when compared to control values). At 4 and 12 mg/kg/day, very slight reductions were noted at different intervals, none of them were statistically significant when compared to control values.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The uterus weight of treated females at the low dose-level of DDAC was similar to that of the control group, while the weight of the gravid uterus of treated females at 12 and 32 mg DDAC/kg/day was slightly lower. These differences were correlated with lower numbers of fetuses.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy findings in the intestines (all treated groups), stomach and gall bladder (high-dose group only) were considered to be related to the treatment with DDAC. No other treatment-related findings were observed. There were no findings at the gross examination of placentae in any females.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

When compared with the control value, the number of dead fetuses was higher (and statistically significant when expressed as a total number) at 32 mg DDAC/kg/day. This difference led to a lower number of live fetuses, and a high mean post-implantation loss. All these mean values for this group were outside CIT background data. It is worth noting that the number of autolyzed fetuses was higher than in the control group.
The high number of autolyzed fetuses recorded in group 4 (32 mg DDAC/kg/day) was due to three litters (E30667, E30669 and E30670) with 5, 13 and 6 autolyzed fetuses. Although the number of dead fetuses recorded in the control group was higher than the CIT background data (mean per female= 0.2), all the differences noted in the litter data described above for the group treated at 32 mg DDAC/kg/day was attributed to treatment with the test item. The modification of these parameters in the low dose group was not considered to be treatment-related in the absence of obvious dose-relationship. The number of live fetuses in the intermediate dose-group and the control group were similar. The percentage of male fetuses and the fetal body weights were unaffected by treatment.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
32 mg DDAC/kg/d:
Evident signs of maternal toxicity (3 mortality), abortion, clinical signs at the end of the gestation; reduction in food consumption and body weight loss) were noted. Necropsy findings concerned the intestines, the stomach and the gall bladder.
Clinical signs were observed in 11/19 animals and consisted of colored urine (5/19), soft feces (4/19), absence feces (3/19), blood in bedding (2/19), and one incidence of loud breathing. One animal showed an emaciated appearance at the end of the study. Autopsy of the surviving animals revealed effects on intestines (8/19: distended with gas, dilated, brownish content), stomach (2/19: dilated, thickened wall, brown/whitish area/deposit), gall bladder (1/19: dilated) and lungs (1/19: brownish foci and reddish areas).

12 mg DDAC/kg/d:
One animal aborted on GD28 after period with absence of feces, weight loss, and autopsy showed dilated intestines and gall bladder reduced in size. One animal was not pregnant. Clinical signs in the remaining surviving animals consisted of absence of feces at end of pregnancy (2/10), loud breathing (1/20) and reddish colored urine from GD22 (1/20). Observations at autopsy: brownish foci in lungs (1/20), accentuated lobular pattern liver (2/10), thickened wall uterus (2/10), and brownish content intestines with liquid in cecum (2/10).

4 mg DDAC/kg/d:
Two animals found dead (GD 10 and 26) both displaying brownish areas/foci in lungs indicating possible aspiration of test item. Clinical signs in the remaining pregnant animals comprised of absence of feces observed at GD29 or from GD28 (3/20). Autopsy showed brownish foci in lungs (1/20), effects on intestines (2/20), and a pale liver (1/20).

Control:
One animal aborted on GD23 after a period of reduced food consumption, abnormal breathing, absence of feces, and blood in bedding. No autopsy findings. Of the remaining pregnant animals at term observations included blood in bedding (2/21), cutaneous lesion (2/20) and immobilized hind limb (1/20). Autopsy showed brownish foci in lungs in 3/20. (Net) body weight changes are difficult to interpret due to the big inter-individual variation. There are also large intra-individual differences between periods of the gestation period. Overall, there seems to be a dose related effect, tending to lower body weights at higher dose levels which corresponding to lower food consumption.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 4 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: effects secondary to local effects

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The fetal body weights were unaffected by treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The percentage of male fetuses were unaffected by treatment.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of fetuses showing external malformations was 1, 0, 2 and 0 distributed in 1, 0, 2 and 0 litters at 0, 4, 12 or 32 mg DDAC/kg/day. This distribution was not considered to be treatment-related. Variations were also distributed in different groups including control group and with no significant dose-related increase.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No dose- or treatment-related increases were noted for any skeletal malformations. At 32 mg DDAC/kg/day, the number of litters with fetuses with incomplete ossification of the sixth sternebrae was higher than control (p<0.05 when expressed as litter incidence). Taking into account that this variation is commonly observed in fetuses of this strain of rabbit, and that incidences were within the CIT background data range, it was not considered to be a consequence of treatment with DDAC. No treatment-related changes were observed at cartilage examination in any of the litters examined. Therefore, it could be considered that the general ossification of fetuses was not affected by treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The only malformations detected at visceral examination were considered to be spontaneous in origin as affecting one fetus in the 0 and 12 mg DDAC/kg/day. The presence of whitish colored fluid in the abdomen (variation) of 5 fetuses from the same litter (5/6 fetuses doe E30667) treated at 32 mg DDAC/kg/day was considered to be most probably related to the test-treatment (statistically significant), as this finding was not observed in any of the fetuses from control group, and both the fetal and litter incidence were outside CIT background data.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 12 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: based on decreased number of live foetuses, increased post-implantation loss and a decreased foetal body weight at 32 mg/kg bw/day

Remarks on result:

other: prenatal effects only seen as unspecific consequence of maternal distress

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

32 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

yes

None

Conclusions:

Based on the results of the read across study, the NOAEL for maternal toxicity and embryotoxic/teratogenic effects of the test substance are considered to be 4 and 12 mg a.i./kg bw/day respectively.

Executive summary:

A study was conducted to evaluate the prenatal developmental toxicity of the read across substance, DDAC (40% active in water) in rabbits by oral gavage route, according to OECD guideline 414 and EPA OPPTS 870.3700, in compliance with GLP. The read across substance at concentrations of 0, 4, 12 and 32 mg a.i./kg bw/day was administered by gavage, to groups of 20 female New Zealand White rabbits from Day 6 to 28 post coitum. All females were submitted to a macroscopic post-mortem examination of the principal thoracic and abdominal organs. A gross evaluation of placentas was also undertaken. Fetuses were examined for skeleton and soft tissue malformations. When given 12 mg a.i./kg bw/day, coloured urine and loud breathing was observed in one female from GD 22 each. Both animals however, showed no findings at autopsy. At 32 mg a.i./kg bw/day, the number of animals showing clinical signs was greater than in the control group, and most of these clinical signs (coloured urine, soft faeces, soiled urogenital area) were recorded at the end of the gestation period, along with an absence of faeces observed for three animals. Presence of blood in the bedding was noted in two females on GD 16 or 22 and one female showed an emaciated appearance on GD 29. There was a big variability in litter data. The low dose group showed higher number of dead fetuses, post implantation loss and lower fetal weight compared to control. On the other hand, the number of live fetuses was even higher than of control. At the mid-dose, observations were comparable to control again. The high-dose group of 32 mg a.i./kg bw/day resulted to an increased post-implantation loss, decrease in live fetuses with a decreased fetal body weight, and high number of dead fetuses. Upon examination of the soft tissue, presence of whitish fluid in the abdomens of 5/6 fetuses (one litter) was observed. No relevant malformations or variations were observed during the fetal evaluation. At lower concentrations, litter parameters were not affected by the test treatment and no evidence of treatment-related findings were noted at skeletal examinations of the fetuses. Overall, there seems to be a dose related effect, tending to lower body weights at higher dose levels which corresponding to lower food consumption related to palatability of the compound in the diet (Chevalier, 2005). Based on the results of the read across study, the NOAEL for maternal toxicity and embryotoxic/teratogenic effects of the test substance are considered to be 4 and 12 mg a.i./kg bw/day respectively. 

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

Pre GLP

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Anglia Laboratory Animals, Alconbury, Huntingdon, Cambridgeshire
- Housing: Opaque plastic cages
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±2 ºC
- Humidity (%): 55± 10 %
- Air changes (per hr): 13 air changes
- Photoperiod : 12 h light and 12 h dark

Route of administration:

dermal

Vehicle:

ethanol

Details on exposure:

TEST SITE
- Area of exposure: Scapular region (4 X4 cm)
- Time intervals for shavings or clipplings: Shaving was repeated when necessary

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL/rat (8, 16.5 or 33 mg/rat) on Day 6-15 of pregnancy
- Constant volume or concentration used: yes

VEHICLE
- Concentration (if solution): 10 % v/v ethanol

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Details on mating procedure:

No data

Duration of treatment / exposure:

From gestation Day 6-15

Frequency of treatment:

Daily

Duration of test:

10 d

Remarks:

Doses / Concentrations:
0.5 mL- 4.4, 6.6, 9.9 %
Basis: nominal conc.

No. of animals per sex per dose:

20 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes


DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Day 1, 3, 6, 10, 17 and 20 of pregnancy


FOOD CONSUMPTION: Yes

WATER CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20

OTHER:
- Foetuses were examined for skeletal and visceral abnormalities

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number and distribution of live young: Yes
- Number and distribution of embryo/foetal deaths : Yes
- Litter weight to calculate the mean pup weight: Yes
- Foetal abnormalities: Yes

Fetal examinations:

- External examinations: Yes:
- Soft tissue examinations: Yes
- Skeletal examinations: Yes

Statistics:

- ANOVA for assessment of intergroup differences in bodyweight, food or water consumption
- Kruskal Wallis test if significant heterogenicity was recorded
- Jonckheerek'test

Indices:

No data

Historical control data:

Yes

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

A dosage related local reaction was recorded in terms of incidence of animals affected, and degree of reaction. In all cases initial reaction was evident within a day of the first administration, reached peak severity around the mid point of the dosing period and stabilized or showed a decline thereafter. Stabilization or decline was frequently associated with scab formation which may have provided a true protection, or obscured reading of erythema and oedema, or both.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Marginal differences from control weight gain during the dosing period were recorded in respect of the higher and lower weight gains respectively associated with low and intermediate dosages of substance.

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Gross pathological findings:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 180.2 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 180.2 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 79.3 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

dermal irritation

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
- Values for the number of live young, embryonic deaths (resorptions) implantations and corpora lutea per dam, litter and mean foetal weight,
were not adversely affected at any dosage level
- No significant differences from concurrent control values in respect of the incidence of malformed or anomalous young or of litters containing affected young
- The types of malformations or anomaly observed were compatible with the types of abnormality recorded among concurrent or historical control values and there was no unusual clustering of a particular type of abnormality in anyone test group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: absence of treatment related adverse effects

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

None

Conclusions:

Under the study conditions, the NOAEL of the test substance for the maternal toxicity and developmental toxicity in rats was determined to be >=180.2 mg a.i./kg bw/day.

Executive summary:

A study was conducted to determine the teratogenicity potential of the test substance, DSDMAC (90.1% active) in rats according to the method similar or equivalent to OECD guideline 414. The test substance was applied topically to 20 female rats on a 4x4 cm area as 0.5 mL per animal throughout gestations days 6 to 15 at concentrations of 0, 4.4, 6.6 and 9.9 %w/v in 10%v/v ethanol in water as vehicle. This resulted to the application of 0, 22, 33 and about 50 mg per animal per day on days 6-15 of the pregnancy [considering mean body weight of animals at day 6 as 250 g, doses are equivalent to 0, 88, 132 and 200 mg/kg bw/day (0, 79.3, 120.7 and 180.2 mg a.i./kg bw/day)]. Test substance induced dose related maternal irritation. A dosage related local reaction was recorded in terms of incidence of animals affected, and degree of reaction. In all cases initial reaction was evident within a day of the first administration, reached peak severity around the mid point of the dosing period and stabilized or showed a decline thereafter. Stabilization or decline was frequently associated with scab formation which may have provided a true protection, or obscured reading of erythema and oedema, or both. There were no systemic signs of reaction, no deaths nor treatment related macroscopic pathological changes in internal organs reported. No marked effects in food or water consumption were observed. No marked or consistently dosage related differences in weight gain were reported. Values for the number of live young, embryonic deaths (resorptions) implantations and corpora lutea per dam, litter and mean foetal weight, were not adversely affected at any dosage level. No significant differences from concurrent control values in respect of the incidence of malformed or anomalous young or of litters containing affected young. The types of malformations or anomaly observed were compatible with the types of abnormality recorded among concurrent or historical control values and there was no unusual clustering of a particular type of abnormality in anyone test group. Under the study conditions, the NOAEL of the test substance for the maternal toxicity and developmental toxicity in rats was determined to be >=180.2 mg a.i./kg bw/day (Palmer, 1983).

Reference 4

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

Pre GLP

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Anglia Laboratory Animals, Alconbury, Huntingdon, Cambridgeshire
- Housing: Opaque plastic cages
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±2 ºC
- Humidity (%): 55± 10 %
- Air changes (per hr): 13 air changes
- Photoperiod : 12 h light and 12 h dark

Route of administration:

dermal

Vehicle:

ethanol

Details on exposure:

TEST SITE
- Area of exposure: Scapular region (4 X4 cm)
- Time intervals for shavings or clipplings: Shaving was repeated when necessary

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL/rat (8, 16.5 or 33 mg/rat) on Day 6-15 of pregnancy
- Constant volume or concentration used: yes

VEHICLE
- Concentration (if solution): 10 % v/v ethanol

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Details on mating procedure:

No data

Duration of treatment / exposure:

From gestation Day 6-15

Frequency of treatment:

Daily

Duration of test:

10 d

Remarks:

Doses / Concentrations:
0.5 mL- 4.4, 6.6, 9.9 %
Basis: nominal conc.

No. of animals per sex per dose:

20 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes


DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Day 1, 3, 6, 10, 17 and 20 of pregnancy


FOOD CONSUMPTION: Yes

WATER CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20

OTHER:
- Foetuses were examined for skeletal and visceral abnormalities

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number and distribution of live young: Yes
- Number and distribution of embryo/foetal deaths : Yes
- Litter weight to calculate the mean pup weight: Yes
- Foetal abnormalities: Yes

Fetal examinations:

- External examinations: Yes:
- Soft tissue examinations: Yes
- Skeletal examinations: Yes

Statistics:

- ANOVA for assessment of intergroup differences in bodyweight, food or water consumption
- Kruskal Wallis test if significant heterogenicity was recorded
- Jonckheerek'test

Indices:

No data

Historical control data:

Yes

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

A dosage related local reaction was recorded in terms of incidence of animals affected, and degree of reaction. In all cases initial reaction was evident within a day of the first administration, reached peak severity around the mid point of the dosing period and stabilized or showed a decline thereafter. Stabilization or decline was frequently associated with scab formation which may have provided a true protection, or obscured reading of erythema and oedema, or both.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Marginal differences from control weight gain during the dosing period were recorded in respect of the higher and lower weight gains respectively associated with low and intermediate dosages of substance.

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Gross pathological findings:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 180.2 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 180.2 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 79.3 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

dermal irritation

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
- Values for the number of live young, embryonic deaths (resorptions) implantations and corpora lutea per dam, litter and mean foetal weight,
were not adversely affected at any dosage level
- No significant differences from concurrent control values in respect of the incidence of malformed or anomalous young or of litters containing affected young
- The types of malformations or anomaly observed were compatible with the types of abnormality recorded among concurrent or historical control values and there was no unusual clustering of a particular type of abnormality in anyone test group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: absence of treatment related adverse effects

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

None

Conclusions:

Based on the results of the read across study, the NOAEL of the test substance for the maternal toxicity and developmental toxicity in rats is considered to be >=180.2 mg a.i./kg bw/day.

Executive summary:

A study was conducted to determine the teratogenicity potential of the read across substance, DSDMAC (90.1% active) in rats according to the method similar or equivalent to OECD guideline 414. The read across substance was applied topically to 20 female rats on a 4x4 cm area as 0.5 mL per animal throughout gestations days 6 to 15 at concentrations of 0, 4.4, 6.6 and 9.9% w/v in 10%v/v ethanol in water as vehicle. This resulted to the application of 0, 22, 33 and about 50 mg per animal per day on days 6-15 of the pregnancy [considering mean body weight of animals at day 6 as 250 g, doses are equivalent to 0, 88, 132 and 200 mg/kg bw/day (0, 79.3, 120.7 and 180.2 mg a.i./kg bw/day)]. Read across substance induced dose related maternal irritation. A dosage related local reaction was recorded in terms of incidence of animals affected, and degree of reaction. In all cases initial reaction was evident within a day of the first administration, reached peak severity around the mid point of the dosing period and stabilized or showed a decline thereafter. Stabilization or decline was frequently associated with scab formation which may have provided a true protection, or obscured reading of erythema and oedema, or both. There were no systemic signs of reaction, no deaths nor treatment related macroscopic pathological changes in internal organs reported. No marked effects in food or water consumption were observed. No marked or consistently dosage related differences in weight gain were reported. Values for the number of live young, embryonic deaths (resorptions) implantations and corpora lutea per dam, litter and mean foetal weight, were not adversely affected at any dosage level. No significant differences from concurrent control values in respect of the incidence of malformed or anomalous young or of litters containing affected young. The types of malformations or anomaly observed were compatible with the types of abnormality recorded among concurrent or historical control values and there was no unusual clustering of a particular type of abnormality in anyone test group (Palmer, 1983). Based on the results of the read across study, the NOAEL of the test substance for the maternal toxicity and developmental toxicity in rats is considered to be >=180.2 mg a.i./kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.8 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Well conducted studies with read across substances; no true systemic effects; systemic NOAELs not used futher for risk assessment.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

180.2 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Well conducted studies available with the read across substance. No true systemic effects; systemic NOAELs not used futher for risk assessment.

Additional information

No development toxicity study could be located on C12-18 DAQ. Therefore, read across studies available with the structurally similar substance DDAC is presented. Both the test and read across substances are di-alkyl dimethyl ammonium chloride compounds. DDAC is structurally the same but only differs in a slightly lower average alkyl chain length. Slightly shorter alkyl chains do not change possible chemical reactivity, but DDAC with a smaller molecule is potentially more bioavailable, and thus is more likely to show effects, therefore representing a worst case. The study with C18 DAQ, which contains long alkyl chain similar to the test substance, has been presented as a supporting data.

Oral exposure

A study was conducted to evaluate the prenatal developmental toxicity of the read across substance, DDAC (40% active in water) in rabbits by oral gavage route, according to OECD guideline 414 and EPA OPPTS 870.3700, in compliance with GLP. The read across substance at concentrations of 0, 4, 12 and 32 mg a.i./kg bw/day was administered by gavage, to groups of 20 female New Zealand White rabbits from Day 6 to 28 post coitum. All females were submitted to a macroscopic post-mortem examination of the principal thoracic and abdominal organs. A gross evaluation of placentas was also undertaken. Fetuses were examined for skeleton and soft tissue malformations. When given 12 mg a.i./kg bw/day, coloured urine and loud breathing was observed in one female from GD 22 each. Both animals however, showed no findings at autopsy. At 32 mg a.i./kg bw/day, the number of animals showing clinical signs was greater than in the control group, and most of these clinical signs (coloured urine, soft faeces, soiled urogenital area) were recorded at the end of the gestation period, along with an absence of faeces observed for three animals. Presence of blood in the bedding was noted in two females on GD 16 or 22 and one female showed an emaciated appearance on GD 29. There was a big variability in litter data. The low dose group showed higher number of dead fetuses, post implantation loss and lower fetal weight compared to control. On the other hand, the number of live fetuses was even higher than of control. At the mid-dose, observations were comparable to control again. The high-dose group of 32 mg a.i./kg bw/day resulted to an increased post-implantation loss, decrease in live fetuses with a decreased fetal body weight, and high number of dead fetuses. Upon examination of the soft tissue, presence of whitish fluid in the abdomens of 5/6 fetuses (one litter) was observed. No relevant malformations or variations were observed during the fetal evaluation. At lower concentrations, litter parameters were not affected by the test treatment and no evidence of treatment-related findings were noted at skeletal examinations of the fetuses. Overall, there seems to be a dose related effect, tending to lower body weights at higher dose levels which corresponding to lower food consumption related to palatability of the compound in the diet (Chevalier, 2005). Based on the results of the read across study, the NOAEL for maternal toxicity and embryotoxic/teratogenic effects of the test substance are considered to be 4 and 12 mg a.i./kg bw/day respectively. 

Further, the DDAC assessment report for Product Type 8 conducted under Directive 98/8/EC (evaluating Competent Authority: Italy, June 2015, attached in Section 13 of the IUCLID dataset) reported two additional studies in rats and rabbits. In these studies, based on local effects, reduced body weight and/or food consumption, which was considered secondary, the maternal NOELs in rats and rabbits were established at 0.8 and 1 mg/kg bw/day respectively. Except for reduced fetal weight in the rabbit study, there no other development toxicity including teratogenicity observed at any dose leading to establishment of respective NOELs at >16.2 mg/kg bw/day in rats and at 3 mg a.i./kg bw/day in rabbits. The lowest NOAEL for maternal toxicity was concluded to be 0.8 mg/kg bw/day, which was not considered relevant for systemic toxicity.

Dermal exposure

A study was conducted to determine the teratogenicity potential of the read across substance, C18 DAQ (90.1% active) in rats according to the method similar or equivalent to OECD guideline 414. The read across substance was applied topically to 20 female rats on a 4x4 cm area as 0.5 mL per animal throughout gestations days 6 to 15 at concentrations of 0, 4.4, 6.6 and 9.9% w/v in 10%v/v ethanol in water as vehicle. This resulted to the application of 0, 22, 33 and about 50 mg per animal per day on days 6-15 of the pregnancy [considering mean body weight of animals at day 6 as 250 g, doses are equivalent to 0, 88, 132 and 200 mg/kg bw/day (0, 79.3, 120.7 and 180.2 mg a.i./kg bw/day)]. Read across substance induced dose related maternal irritation. A dosage related local reaction was recorded in terms of incidence of animals affected, and degree of reaction. In all cases initial reaction was evident within a day of the first administration, reached peak severity around the mid point of the dosing period and stabilized or showed a decline thereafter. Stabilization or decline was frequently associated with scab formation which may have provided a true protection, or obscured reading of erythema and oedema, or both. There were no systemic signs of reaction, no deaths nor treatment related macroscopic pathological changes in internal organs reported. No marked effects in food or water consumption were observed. No marked or consistently dosage related differences in weight gain were reported. Values for the number of live young, embryonic deaths (resorptions) implantations and corpora lutea per dam, litter and mean foetal weight, were not adversely affected at any dosage level. No significant differences from concurrent control values in respect of the incidence of malformed or anomalous young or of litters containing affected young. The types of malformations or anomaly observed were compatible with the types of abnormality recorded among concurrent or historical control values and there was no unusual clustering of a particular type of abnormality in anyone test group (Palmer, 1983). Based on the results of the read across study, the NOAEL of the test substance for the maternal toxicity and developmental toxicity in rats is considered to be ≥180.2 mg a.i./kg bw/day.

Overall, the available oral and dermal pre-natal development toxicity studies with the read across substances in rats and rabbits, indicate no concern for development toxicity. In line with the biocides assessment report, the maternal NOAELs from the pre-natal studies have not been considered further for risk assessment. 

Justification for classification or non-classification

Based on the results of the read across two-generation reproductive toxicity and pre-natal development toxicity studies in rats and rabbits, the test substance does not warrant a classification according to EU CLP criteria (Regulation 1272/2008/EC).