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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: feed
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
- Exposure period: about 18 wk.
P0 and P1:
Premating exposure period (males): 10 wk.
Premating exposure period (females): 10 wk.
- Duration of test: P0 pre-mating 10 wk, until F2 weaning.
Frequency of treatment:
Continuously
Details on study schedule:
Groups of 25 male and 25 female Sprague-Dawley rats were administered the test substance (purity 49.9%) at levels of 0, 500, 2000 or 4000 ppm in their diet over a period of 10 wk before mating, 2 wk during mating, and until after weaning of the pups (total period 18 wk). From every litter one or two pups were selected to again obtain 25 animals per sex and dose group for the F1 generation. These animals were similarly treated as the parent (P1) animals throughout premating, mating, pregnancy until sacrifice, after weaning of F2 progeny.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations: 0, 500, 2000 or 4000 ppm (equivalent to 250, 1000 and 2000 a.i. ppm) of test substance
Remarks:
F0 generation - 16-25, 61-101, 123-208 mg/kg bw/day (equivalent to 8-13, 31-51, 62-104 mg a.i./kg bw/day); F1 generation - 24-34, 96-123, 202-252 mg/kg bw/day (equivalent to 12-17, 48-62, 101-126 mg a.i./kg bw/day)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
For details, kindly refer to the attached background material section of the IUCLID.

Examinations

Parental animals: Observations and examinations:
- Examination of P0 and P1 generation:
Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals. Males and females were paired for a 2-wk period, until mating was obtained. The P1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded.
- During lactation, the pups (F1 generation) were observed daily for survival and clinical signs; body weight was recorded at designated intervals; the sex-ratio was recorded. On Day 4 post-partum, the size of each litter was adjusted to obtain eight pups per litter (four males and four females).
- Reflex development was assessed at designated time-points.
Sperm parameters (parental animals):
Epididymal and testicular sperm parameters were evaluated for both P0 and P1 males.
Litter observations:
Examination of F1/P1 generation:
- On Day 22 post-partum, one male and one female pup per litter were selected to constitute the F1/P1 generation, which comprised 25 males and 25 females per group. The F1/P1 animals were observed daily for clinical signs and mortality. Body weight and food consumption were recorded once a week. Sexual development of both males and females was assessed.
- Neurobehavioural tests were conducted at designated intervals to assess auditory and visual functions. Spontaneous locomotor activity was also evaluated when the animals were between 7 and 8 wk old.
- After sexual maturity, F1 male and F1 female animals were paired. The F1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded. During lactation, the pups (F2 generation) were observed daily for survival and clinical signs; body weight was recorded at designated intervals; the sex-ratio was recorded. On day 4 post-partum, the size of each litter was adjusted to obtain eight pups per litter (four males and four females).
- Reflex development was assessed at designated time-points.
Postmortem examinations (parental animals):
Terminal examination of P0 and P1 animals:
- After weaning of their respective progeny, P0 and P1 parent males and females were sacrificed. Designated organs were weighed for P0 and P1 parents, as well as brain, spleen and thymus of one pup per sex per litter of each generation.
- Epididymal and testicular sperm parameters were evaluated for both P0 and P1 males.
- A macroscopic post-mortem examination was performed on all P0 and P1 parent males and females. Any pups which died or were killed prematurely during the lactation period were also submitted for a macroscopic post-mortem examination. Macroscopic lesions, reproductive organs, adrenals and pituitary glands were sampled in all parent animals. A microscopic examination was performed on macroscopic lesions, reproductive organs, adrenals, and pituitary glands of all P0 and P1 parents of the control and high dose groups.
- Particularly detailed histopathological examinations were performed for the ovaries and the testes.
Postmortem examinations (offspring):
- A macroscopic post-mortem examination was performed on three pups per sex and per litter of each P0 and P1 (F1 and F2 generation) females killed at weaning. Any pups which died or were killed prematurely during the lactation period were also submitted for a macroscopic post-mortem examination. In all pups, the macroscopic lesions were preserved.
Statistics:
For details, kindly refer to the attached background material section of the IUCLID.
Reproductive indices:
For details, kindly refer to the attached background material section of the IUCLID.
Offspring viability indices:
For details, kindly refer to the attached background material section of the IUCLID.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, a slightly lower mean body weight gain was recorded during most of the dosing period in both parental males and females (and was associated with reduced liver weights).
- At 1000 and 250 ppm, a marginally to slightly lower body weight gain was noted over all the dosing period for the males.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, a slightly to moderately lower mean food consumption was recorded during most of the dosing period in both parental males and females.
- At 1000 and 250 ppm, a marginally to slightly lower mean food consumption was noted over all the dosing period for the males.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- At 2000 and 1000 ppm, no effects were noted at histopathological examination of sexual organs.

Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- At 2000 and 1000 ppm, no effects were noted on sperm parameters.

Reproductive performance:
no effects observed
Description (incidence and severity):
- At 2000 ppm, slightly lower pup body weight was observed (with lower spleen weights).
- At 1000 ppm, no effects were observed on parental fertility as assessed by normal mating, gestation and delivery.
- At 250 ppm, no effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny.

Details on results (P0)

As lower food consumption is known to occur due to palatability of compound. As no other substance related effects were seen than can be attributed to lower food intake, the level of 250 ppm test substance in the diet, corresponding to 16-25 and 24-31 mg/kg bw/day, should be regarded as NOAEL for P0 and P1generation respectively.
For further details, see under Any other information on results incl. tables.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
ca. 250 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: equivalent to 16-25 mg/kg bw/day (8-12.5 mg a.i./kg bw/day)
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
ca. 1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects on mating behaviour, fertility and gestation of each generation
Remarks on result:
other: equivalent to 61-101 mg/kg bw/day (30.5-50.5 mg a.i./kg bw/day)

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, a slightly to moderately lower mean body weight gain was recorded during most of the dosing period in both parental males and females (and was associated with reduced liver weights).
- At 1000 ppm, a marginally to slightly lower mean body weight gain was noted.
- At 250 ppm, a marginally to slightly lower mean body weight gain was observed in both sexes. This was associated with lower liver weights of parental males and females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, a slightly to moderately lower mean food consumption was recorded during most of the dosing period in both parental males and females.
- At 1000 and 250 ppm, a marginally to slightly lower mean food consumption was noted over all the dosing period in both sexes.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, reduced liver weights were observed.
- At 1000 and 250 ppm, lower liver weights in parental animals were recorded.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, necropsy of these animals revealed dilation of the cecum, colon or ileum in some animals (more marked in F0 parents).
- At 1000 ppm, necropsy of parents revealed dilatation of the cecum in a single animal. 
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- At 2000 and 1000 ppm, no effects were noted at histopathological examination of sexual organs.
Histopathological findings: neoplastic:
not examined
Details on results:
See under Any other information on results incl. tables for details on results.

Reproductive function / performance (P1)

Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- At 2000 and 1000 ppm, no effects were noted on sperm parameters.

Reproductive performance:
no effects observed
Description (incidence and severity):
- At 2000 ppm, slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of P1 females) and a delay in sexual development. Lower spleen weights were also noted for each progeny.
- At 1000 ppm, no effects were noted on parental fertility as assessed by normal mating, gestation and delivery.
- At 250 ppm, no effects were noted on mating, fertility, gestation, fecundity or delivery and development of their progeny.

Details on results (P1)

As lower food consumption is known to occur due to palatability of compound. As no other substance related effects were seen than can be attributed to lower food intake, the level of 250 ppm test substance in the diet, corresponding to 16-25 and 24-31 mg/kg bw/day, should be regarded as NOAEL for P0 and P1generation.

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
ca. 250 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: equivalent to 24-31 mg/kg bw/day (12-15.5 mg a.i./kg bw/day)
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
ca. 1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects on mating behaviour, fertility and gestation of each generation
Remarks on result:
other: corresponding to 96-123 mg/kg bw/day (48-61.5 mg a.i./kg bw/day)

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, the litter size at birth was reduced. 

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, slightly reduced pup body weight was observed. Pup weight gain was also slightly lower during lactation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, lower spleen weights were noted.
- At 1000 ppm, except for a marginally lower spleen weight of the progeny, no other effects were noted on their development.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

See under Any other information on results incl. tables for details on results.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Generation:
F1
Effect level:
ca. 250 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: a marginally to slightly lower mean food consumption and body weight gain and liver weights
Remarks on result:
other: corresponding to 24-31 mg/kg bw/day (12-15.5 mg a.i./kg bw/day)
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
ca. 1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: mean age of preputial separation and vaginal opening was delayed due to reduced food intake
Remarks on result:
other: corresponding to 96-123 mg/kg bw/day (48-61.5 mg a.i./kg bw/day)

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, the litter size at birth were reduced (as a consequence of lower number of implantation sites of F1 parent females).
- At 1000 ppm, no effects were seen in F2 offspring regarding pup survival until weaning.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, slightly lower pup body weight was observed. The pup weight gain was slightly reduced during lactation, 
- At 1000 ppm, no effects were seen regarding pup development.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, lower spleen weights were noted.
- At 1000 ppm, except for a marginally lower spleen weight, no other effects were noted on their development.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 2000 ppm, dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females.
At 1000 ppm, no effects were seen regarding pup development and after sacrifice at weaning.
Histopathological findings:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Remarks:
general / developmental toxicity
Generation:
F2
Effect level:
1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
mortality
other: based on lower pup weights, lower pup weight gain during lactation, and upon necropsy dilatation of the cecum with feces was observed in 4/25 males and 2/25 females
Remarks on result:
other: corresponding to 96-123 mg/kg bw/day (48-61.5 mg a.i./kg bw/day)

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

The mean achieved dosages of the test substance for the dose-levels of 250, 1000 and 2000 ppm of test substance were as follows:
F0 generation
- males (Days 1 to 106): 16, 61 and 123 mg/kg bw/day, respectively,
- females:

during premating period (Days 1 to 71): 19, 74 and 154 mg/kg bw/day, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 18, 69 and 145 mg/kg bw/day, respectively,
    during lactation period (Days 1 to 21 p.p.): 37, 159 and 326 mg/kg bw/day, respectively.
F1 generation
- males (Days 1 to 120): 24, 96 and 202 mg/kg bw/day, respectively,
- females:
    during premating period (Days 1 to 64): 32, 127 and 269 mg/kg bw/day, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 21, 83 and 164 mg/kg bw/day, respectively,
    during lactation period (Days 1 to 21 p.p.): 41, 162 and 323 mg/kg bw/day, respectively.

The actual intake of test substance for both males and females given 250, 1000 and 2000 ppm throughout the study is approximately 16-25, 61-101 and 123-208 mg/kg bw/day, respectively  for the F0 generation and 24-31, 96-123 and 202-252 mg/kg bw/day for the F1 generation.

Details on results:

Parent males (F0):

No effects:mortality, clinical signs, mating and fertility indices, seminology parameters, macroscopic post-mortem examination and histopathology.

 

Body weight:

 

During the first week of dosing, the body weight gains were slightly low at the low and mid dose-levels (-13% and -11%, p<0.01, p<0.05, respectively) and moderately low at the high dose-level (days 1 to 8, -24%, p<0.001). This effect on body weight gain was observed over all the dosing period (days 1 to 106, - 11%, p<0.01; -8%, non-statistically significant and -20%, p<0.001, at 250, 1000 and 2000 ppm, respectively).

At all dose-levels, when compared to controls, body weights were marginally lower for the low and mid dose-levels (between -5 and -7%, p<0.01) and slightly lower for the high dose-level during most of the dosing period (-13%, p<0.001).

 

Food consumption:the food consumption during the premating period was marginally low at 250 and 1000 ppm reaching statistical significance during the first weeks of dosing (weeks 1, 3 and 4) and sporadically during 5 or 7 weeks (between -7% and -10%, p< 0.01, p<0.001). This effect was more marked at 2000 ppm and was statistically significant during the whole dosing period (between -7 and -14%, p<0.01, p<0.001).

 

Organ weights (expressed in %)

Lower absolute and body weight-related liver weights in the males given 1000 or 2000 ppm were statistically significant. In addition, the absolute value of the differences observed was of higher magnitude than that observed for the differences in body weights and the absolute and body weight-related differences were dose-related in the males. These differences were considered to be due to the treatment with the test substance, C12-16 ADBAC. It is worth mentioning that similar differences in liver weight without histomorphological changes were observed in the previous 90-day study.

 

Parent females F0

No effects:mortality, clinical signs, mating and fertility indices, estrous cycle, pregnancy and parturition data (implantation, fecundity, post-implantation, and neo-natal losses, gestation and delivery parameters) and histopathology.

Body weight:The body weight gain was unaffected by treatment at 250 and 1000 ppm. A moderately, statistically significantly, low body weight gain was observed at 2000 ppm during the first and the fourth week of premating (-31%, p<0.001 and -38%, p<0.01, respectively) and over all pregnancy (-13%, p<0.01) while it increased during the lactation period (24 g vs. 0 g, p<0.001). At this latter dose-level, body weights were lower during most of the dosing period (ranging between -4 and -9%, p<0.05, p<0.01) but returned to values comparable to controls at the end of the lactation period (316 g vs. 325 g in controls).

Food consumption:

The food consumption was unaffected by the treatment at 250 and 1000 ppm while it was slightly reduced at 2000 ppm, reaching statistical significance during the first week of dosing (-11%, p<0.001), during 2 weeks of the premating period (days 22 to 29 and days 64 to 71, -5% p<0.01) and during 2 weeks of the pregnancy period (days 0 to 7 p.c., -4%, p<0.05 and days 7 to 14,

-8%, p<0.001).

During the first week of the lactation period, group mean food consumption was slightly increased in the group given 2000 ppm and values were thereafter comparable to controls until the end of lactation.

 

Organ weights (expressed in %)

Lower absolute and body weight-related liver weights in females given 1000 or 2000 ppm were statistically significant. In addition, the absolute value of the differences observed was of higher magnitude than that observed for the differences in body weights. These differences were considered to be due to the treatment with the test item BKC. It is worth mentioning that similar differences in liver weight without histomorphological changes were observed in the previous 90-day study.

Macroscopic post-mortem examination

Some segments of the intestinal tract were distended with faeces at necropsy with higher incidence in the females given 2000 ppm, compared with their respective controls.

Although no microscopic finding could be correlated, considering the higher incidence of this finding in the test item-treated females compared with their respective controls, distension of intestinal tract was considered to be related to treatment with the test item. This included:

-         ileum in 1/25 females given 1000 ppm and 5/25 females given 2000 ppm vs. none control female,

-         coecum in 1/25 females given 250 ppm, 8/25 given 1000 ppm and 12/25 females given 2000 ppm vs. 2/25 control females,

-         colon in 1/25 females given 1000 ppm and 2/25 females given 2000 ppm vs. none control female.

F1 males

No effects:mortality, clinical signs, neurobehavioral tests, mating and fertility indices, seminology parameters and histopathology.

Body weight:On day 1 (post-natal day 22), the body weight was slightly lower at 2000 ppm (-13%, p<0.001) while it was minimally, but not statistically significantly,

affected at 1000 ppm (-5%) and was similar to controls at 250 ppm. During the whole dosing period, the body weight was generally slightly (for the low and mid-dose levels) to moderately (for the high dose-level), but statistically significantly, lower. The body weight gain was statistically significantly lower during the two first weeks of dosing at all dose-levels and remained markedly lower over all the dosing period at the high dose-level (days 1 to 120, - 18%,p<0.001).

Food consumption:

In the males, group mean food consumption was slightly to moderately lower at all dose-levels (ranging between -7 and -21%, p<0.05, p<0.01, p<0.001) reaching statistical significance during many periods at the low and mid dose- levels and during all recorded periods at the high dose-level (p<0.01, p<0.001).

Sexual development

The mean age at preputial separation (cleavage of the balanopreputial gland) was unaffected by the treatment at 250 and 1000 ppm (i.e occurred at 49.6 and

50.1 day old, respectively vs. 47.8 day old in controls) while, when compared to the male control group, a significant difference was observed in males given 2000 ppm (52.80 ± 6.02 vs. 47.76 ± 2.62 days, p<0.0019). It was not statistically significant when body weight was considered as a covariate, which suggests that this effect was the consequence of a lower body weight rather than a direct effect on sexual development.

Organ weights (expressed in %)

Lower absolute and/or body weight-related liver weights in the males given 250, 1000 or 2000 ppm were observed, with statistical significance. In addition, the absolute value of the differences observed was of higher magnitude than that observed for the differences in body weights. These differences were therefore considered to be due to treatment with the test item, BKC. It is worth mentioning that similar differences in liver weight without histomorphological changes were observed in the previous 90-day study and similar differences in liver weights were also noted in the F0 generation.

Macroscopic post-mortem examination

The coecum was distended with faeces in 1/25 males given 1000 ppm and in 4/25 males given 2000 ppm.

Although no microscopic finding could be correlated (at least for the macroscopic abnormalities which were examined), considering the previous observations made in the intestinal tract in the females of the F0 generation, this finding reported in a few test item-treated animals of both sexes, was considered to be most probably related to treatment with the test item.

 

F1 females

No effects:mortality, clinical signs, neurobehavioral tests, mating and fertility indices, oestrous cycle, pregnancy and parturition data (with the exception of number of implantation sites and number of live foetuses), and histopathology.

Body weight:On day 1 (post-natal day 22), the body weight was statistically significantly lower at 1000 and 2000 ppm (-6%, p<0.005 and -13%, p<0.001, respectively) and remained lower over all the premating and pregnancy periods until the second week of lactation. The body weight gain was marginally reduced (-9%, not statistically significant and -15% p<0.001, for the low and mid-dose-levels, respectively) to markedly reduced (-41%, p<0.001 for the high dose-level) during the first week of premating and over the pregnancy periods (-10%, -11%, -19%, respectively, p<0.01 or p<0.001) while it was increased when compared to controls during the lactation period.

Food consumption:

In the females, group mean food consumption was unaffected by the treatment at the low and mid dose-groups while it was lower at 2000 ppm during premating and pregnancy, reaching statistical significance during most of the recorded periods (ranging between -9 and -14%, p<0.01, p<0.001). No effect was noted during the lactation period.

Sexual development

The mean age for vaginal opening was unaffected by the treatment, i.e. occurred on days 34.1, 33.7, 35.4 for pups from the control, 250 and 1000 ppm groups, respectively while it was delayed for 4 days at 2000 ppm.

When compared to the female control group, a significant difference was observed in females given 2000 ppm (38.40 ± 5.16 vs. 34.12 ± 2.19 in controls p<0.0001), however it was not statistically significant when body weight was considered as a covariate which suggests that this effect was the consequence of a lower body weight rather than a direct effect on sexual development.

Furthermore, animals that had values outside the range of individual control values had the lowest weight at sexual maturity which confirm that this effect was the consequence of reduced body weight and not a direct effect on sexual development.

Pregnancy and parturition data

The statistically significantly lower number of implantation sites and the lower number of live foetuses (-13%, p<0.001 for both parameters) in females given 2000 ppm when compared to controls, were considered to be treatment- related, although these values were within the range of historical control data.

Organ weights (expressed in %)

Lower absolute and/or body weight-related liver weights in the females given 250, 1000 or 2000 ppm were observed, with statistical significance and dose- relationship in the females. In addition, the absolute value of the differences observed was of higher magnitude than that observed for the differences in body weights. These differences were therefore considered to be due to treatment with the test item, BKC. It is worth mentioning that similar differences in liver weight without histomorphological changes were observed in the previous 90-day study and similar differences in liver weights were also noted in the F0 generation.

Macroscopic post-mortem examination

The cecum was distended with feces in 2/25 females given 2000 ppm. Although no microscopic finding could be correlated (at least for the macroscopic abnormalities which were examined), considering the previous observations made in the intestinal tract in the females of the F0 generation, this finding reported in a few test item-treated animals of both sexes, was considered to be most probably related to treatment with the test item.

 

F2 males

No effects:viability and lactation indices, clinical signs, gross external abnormalities, assessment of reflex development (surface righting, cliff avoidance and air righting) and macroscopic post-mortem examination.

Body weight:No effects on body weight or body weight gain were noted at 250 and 1000 ppm while slightly lower body weight gain was observed at 2000 ppm between day 4 and day 21 post-partum (-8%, p<0.05).

Although this difference in body weight gain was slight, it was considered to be possibly related to the treatment.

Organ weights (expressed in %)

Considering that the differences in absolute and body weight-related weights of the spleen of the animals given 1000 or 2000 ppm were dose-related in the two sexes and that the absolute value of the difference in the absolute spleen weight was marginally higher than that observed for the difference in body weight at necropsy, a relationship to treatment could not be excluded, as similar changes were observed in the spleen weights of the pups of the F1 generation.

Macroscopic post-mortem examination

The cecum was distended with feces in 2/25 females given 2000 ppm. Although no microscopic finding could be correlated (at least for the macroscopic abnormalities which were examined), considering the previous observations made in the intestinal tract in the females of the F0 generation, this finding reported in a few test item-treated animals of both sexes, was considered to be most probably related to treatment with the test item.

F2 females

No effects:viability and lactation indices, clinical signs, gross external abnormalities, assessment of reflex and physical development (surface righting, cliff avoidance and air righting) and macroscopic post-mortem examination.

Body weight:No effects on body weight or body weight gain were noted at 250 and 1000 ppm while slightly lower body weight gain was observed at 2000 ppm between day 4 and day 21 post-partum (-8%, p<0.05).

Although this difference in body weight gain was slight, it was considered to be possibly related to the treatment.

Organ weights (expressed in %)

Considering that the differences in absolute and body weight-related weights of the spleen of the animals given 1000 or 2000 ppm were dose-related in the two sexes and that the absolute value of the difference in the absolute spleen weight was marginally higher than that observed for the difference in body weight at necropsy, a relationship to treatment could not be excluded, as similar changes were observed in the spleen weights of the pups of the F1 generation.

Offspring of F0 parents (F1 pups)

No effects:viability and lactation indices, clinical signs and gross external abnormalities, assessment of reflex development and macroscopic post-mortem examination.

Body weight:At birth, pup body weights were unaffected by the treatment at all dose-levels. During the lactation period, pup body weight and body weight gain were not affected at 250 ppm.

At 1000 and 2000 ppm, pup body weight gains were statistically significantly lower during the last week of lactation (-9%, p<0.05 and -11%, p<0.01) while body weight was statistically significantly lower on day 21 post-partum for the high-dose only (-7%, p<0.05). This effect was considered to be most probably related to pup food intake (which normally started during the last week of lactation) and was therefore considered to be related to the treatment.

Organ weights (pups sacrificed at weaning)

Dose-related lower absolute and body weight-related spleen weights were observed in the male and female pups given 1000 or 2000 ppm. This achieved statistical significance for the absolute weight for the animals given 2000 ppm. Almost all the individual weights were within the control range for the test item-treated animals; however they were gathered at the lower limit of the range, especially at the high dose-level.

For the differences in spleen weights, a relationship to treatment with the test item cannot be ruled out, as the same trend was observed in the pups of the F1 generation. However, they might be stress-related, considering the young age of the animals and/or the consequence, at least in part, of the decrease in body weight.

 

Results for F0 and F1 generation summarised according to dose groups:

The following results were obtained:

F0 generation (during premating, mating, pregnancy and lactation until day 21 post-partum of F0 parents and post-natal day 21 for their progeny)

Group 4: 2000 ppm

-        no treatment related clinical signs or mortality were noted,

-        for the parent males, over all the dosing period, statistically significantly low food consumption (between -7 and -14%, p<0.01 and p<0.001), low body weights (-6 and -13%, p<0.001) and reduced body weight gains (- 20%, p<0.001) were noted,

-        for the parent females, the food consumption was reduced during the first week of the premating period (-11%, p<0.001) and during the 2 first weeks of pregnancy (-9% and -8%, respectively). This was associated with lower body weight gain during the first week of the premating period (-31%, p<0.001), during the pregnancy period (-13%, p<0.01) and lower body weights over all the premating and pregnancy periods (between -4% and -9%, p<0.05, p<0.01). The food consumption and the body weight gains were slightly increased during the first week of lactation,

-        no effect on mating, male and female fertility (including estrous cycle and seminology parameters) or on the progress of pregnancy or delivery,

-        no effect on pup survival (F1 generation pups) while reduced pup body weight gain was noted during the last week of lactation (-11%, p<0.01),

-        necropsy of parents revealed dilatation of ileum in 5/25 females, dilatation of cecum in 12/25 females and of the colon in 2/25 females,

-        lower absolute and body weight-related liver weights in parental males (respectively, -23%, -11%, p<0.01) and females (respectively, -16 % and

-        11%, p<0.01) and lower absolute and relative spleen weights of their progeny (respectively, -16%, p<0.05 and -9% for the males, -14%, p<0.05 and -8% for the females),

-        no histopathological findings were noted at microscopic examination of  the different organs (including reproductive organs).

 

Group 2 and 3: 250 and 1000 ppm

-        no treatment related deaths or clinical signs were observed,

-        for the parent males, the food consumption was marginally low during the first week of dosing and sporadically during 4 or 5 weeks (between -7% and -10%, p<0.01, p<0.001) and was associated with a slightly low body weight gain during the first week of dosing (-13% and -11%, p<0.01, p<0.05, respectively) and over all the dosing period (-11%, p<0.05; -8%, respectively at both dose-levels),

-        for the parent females, the food consumption and body weight gains were unaffected by the treatment,

-        no effect on mating, male and female fertility (including estrous cycle and seminology parameters), gestation, fecundity or delivery,

-        no effect on progeny survival from delivery until weaning (F1 generation pups),

-        necropsy of parental males and females given 1000 ppm revealed dilatation of the cecum with feces in 8/25 females and dilatation of the ileum and the colon in 1/25 females,

-        at 1000 ppm, there were lower absolute and body weight-related liver weights of parental males (respectively, -12%, p<0.05 and -7%, p<0.05) and females (-14% and -11%, p<0.01) and marginal, non-statistically significant, lower absolute and body weight-related progeny spleen weights (-7% and -4% for the males and -9% and -6% for the females),

-        at 250 ppm, no effect was noted on parents or progeny‟ organ weights,

-        no histopathological findings were noted at microscopic examination of  the different parental organs (including reproductive organs) at either dose-level.

 

F1 generation (from post-natal day 22, during mating, pregnancy and lactation until day 21 post-partum)

Group 4: 2000 ppm

-        no treatment-related clinical signs or mortality in parental males or females,

-        for the males, the food consumption was lower during the whole dosing period (between -12 and -21%, p<0.0001) and the body weight and body weight gain were moderately lower over all the dosing period (-18%, p<0.001),

-        for the females, reduced food consumption was noted during the premating and pregnancy periods (ranging between -9% and -14%, p<0.01 and p<0.001). This was associated with lower body weight from the first day of the premating period until the second week of lactation and lower body weight gain during the first week of premating (-41%, p<0.001) and over all the premating and pregnancyperiods,

-        the mean age of preputial separation and vaginal opening was delayed   for approximately 5 and 4 days, respectively (52.8 vs. 47.76 day old for preputial separation and 38.4 vs. 34.12 day old for vaginal opening) as a consequence of lower body weight, while no effect was noted on their locomotor activity or on neurobehavioralparameters,

-        no effects were noted on mating or parental fertility (including number of pregnant females with liveborn, estrous cycle and seminology parameters), however, the number of implantation sites and the litter size at birth were reduced (13 vs. 14.9 and 12.4 vs. 14.2, p<0.01, respectively),

-        the pup weight gain of the progeny of the F1 generation (F2 generation pups) was slightly lower during the lactation period (-8%,p<0.05),

-        necropsy of the F1 generation revealed dilatation of the cecum with feces in 4/25 males and 2/25females,

-        lower absolute and body weight-related liver weights of parental males (respectively, -28% and -12%, p<0.01) and females (respectively, -25% and -18%, p<0.01) and decreased absolute and relative spleen weights of their progeny (-23%, p<0.01 and -10% for the males, -26% and -14%, p<0.01 for the females),

-        no histopathological findings were noted at microscopic examination of  the different organs (including reproductiveorgans).

 

Group 3: 1000 ppm

-        no treatment-related clinical signs in parental males andfemales,

-        for the males, the food consumption was statistically significantly lower during many dosing periods (between -6% and -11%, p<0.05, p<0.01, p<0.001) and was associated with a slightly lower body weight and body weight gain over all the dosing period (-7%),

-        for the females, the body weight and food consumption were slightly lower during the first week of premating and during pregnancy (body weight: -15%, p<0.001 and -11%, p<0.01, food consumption: between -9 and -14%, p<0.01,p<0.001),

-        no effect on mating, parental fertility (including estrous cycle and seminology parameters), gestation, fecundity ordelivery,

-        no effect on pup development and progeny survival from delivery until weaning,

-        no effect on locomotor activity and neurobehavioural parameters of the progeny,

-        necropsy of the parents revealed dilatation of coecum with faeces in 1/25 males,

-        lower absolute and body weight-related liver weights of parental males (respectively, -13%, p<0.05 and -6%, not statistically significant) and females (respectively, -17% and -14%, p<0.01) and marginally lower absolute and body weight-related spleen weight of their progeny (-3% for the males and -11 and -10%, respectively for the females),

-        no histopathological findings were noted at microscopic examination of  the different organs (including reproductive organs).

Group 2: 250 ppm

-        no effects at clinical examination of the male and female parents,

-        for the males, body weight and body weight gain were slightly, but statistically significantly, lower during most of the dosing period (-9%, p<0.05) while the food consumption was transiently lower,

-        for the females, the body weight gain was slightly lower during pregnancy (days 0 to 20 p.c., -10%, p<0.01) while no effect was noted on food consumption,

-        no effect on mating, male and female parental fertility (including estrous cycle and seminology parameters), gestation, fecundity or delivery,

-        no effect on sexual development or on neurobehavioral parameters,

-        no effect on the development of the progeny from delivery until weaning,

-        no macroscopic findings in parental males and females or their progeny,

-        lower absolute and body weight-related liver weights of parental males (respectively, -14%, p<0.05 and -6%, not statistically significant) and females (respectively, -14% and -12%, p<0.01) but no effect was observed on organ weights of the progeny,

-        no microscopic findings for male and female parents.

Applicant's summary and conclusion

Conclusions:
Based on the results of the read across study, the NOAELs for parental, reproductive and development toxicity were established at 250, 2000 and 1000 ppm respectively.
Executive summary:

A two-generation study was conducted to determine the toxicity to reproduction of the read across substance, C12 -16 ADBAC (49.9% 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, 250, 1000 and 2000 ppm (i.e., equivalent to 0, 16-25, 61-101 and 123- 208 mg a.i./kg bw/day, respectively for the F0 generation and 24-31, 96-123 and 202-252 mg a.i./kg bw/day for the F1 generation respectively). Doses were administered before and throughout mating and gestation until the end of the lactation period in both F0 and F1 generations.

At 2000 ppm, a slightly to moderately lower mean food consumption and mean body weight gain were recorded during most of the dosing period in both parental males and females of the two generations and was associated with reduced liver weights. Necropsy of these animals (parents of both generations) revealed dilatation of the cecum, colon or ileum in some animals (more marked in F0 parents). No effects were noted on sperm parameters or on histopathological examination of sexual organs. Slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of F1 parent females) and a delay in sexual development. Lower spleen weights were also noted for each progeny.

At 1000 ppm, P0 (males) and P1 (both sexes) showed a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. Necropsy of parents of both generations revealed dilatation of the cecum in some animals of the F0 generation and in a single animal of the F1 generation. This was associated with lower liver weights in parental animals of both generations. No effects were noted on parental fertility as assessed by normal mating, gestation and delivery and, particularly, there were no effects on sperm parameters or at histopathological examination of sexual organs. Except for a marginally lower spleen weight of the progeny of each generation, no other effects were noted on their development.

At 250 ppm (corresponding to approximately 16-25 mg a.i./kg bw/day for F0 males and females and 24-31 mg a.i./kg bw/day for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. This was associated in the F1 generation with lower liver weights of parental males and females. No effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny.

The lower food intake, which was most pronounced in period of start of intake as well in F0 as in F1 animals, indicated that the intake was influenced by palatability of the compound and not by a toxic mechanism. At 250 ppm, the small effects on body weight, body weight changes and absolute and relative organ weights were considered to be resulting from reduced food intake. Similar was the case for the 1000 ppm group. As this was not considered to be an adverse toxic effect from exposure to the read across substance, 250 ppm was considered to be the NOAEL for parental systemic toxicity. The NAOEL for mating behaviour, fertility and gestation of each generation was considered to be 2000 ppm. The marginally lower absolute and body weight-related spleen weight of the F2 progeny at 1000 ppm was also considered to be indirectly caused by the lower food intake of F1 parents, and therefore was not considered to be an adverse toxic effect from exposure to the read across substance. Therefore, the NOAEL for development, growth and survival of each progeny was considered to be at 1000 ppm. Under the conditions of the study, the NOAELs for parental, reproductive and development toxicity were established at 250, 2000 and 1000 ppm respectively (Foulon, 2008).