Hexanoic acid, 6-[(3,5,5-trimethyl-1-oxohexyl)amino]-, compd. with 2,2',2''-nitrilotris[ethanol] (1:1)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was designed similar to guideline OECD 415 and was performed according to GLP guidelines, but was designated mainly to select dose levels for a subsequent definite reproduction study. No histological examination of the reproductive or other organs were performed.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

It is hypothesized that the target chemical and the following chemical as source chemicals should exhibit comparable toxicity profiles:

• 3,5,5-trimethylhexanoic acid

It is proposed to use the toxicity data of the mentioned source chemicals to fulfill the data requirement for the target chemical on the human health endpoint 'reproduction toxicity'. 3,5,5-trimethylhexanoic acid is a presumed metabolite.

The underlying scientific rationale for the use of 3,5,5-trimethylhexanoic acid as source chemical is based on the metabolism consideration. Upon absorption, the target chemical is expected to undergo a degradation process, resulting in the systemic release of 3,5,5-trimethylhexanoic acid, thereby providing the justification for the read-across especially for the mid- and long term toxicities such as repeated dose toxicity and reproduction toxicity.

The proposed approach applies for all exposure routes (oral/dermal/inhalation), because both the target chemical and source chemicals are expected to be bioavailable by all exposure routes and the systemic release of the presumed metabolite is less dependent on exposure route.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

Target chemical:
6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol; CAS: 85702-79-0

Source chemicals:

3,5,5´-trimethylhexanoic acid; CAS: 3302-10-1

The target chemical is a mono-substituent substance, the analytical purity being >99%. The source chemical is the raw materials (3,5,5´-trimethylhexanoic acid) of the target chemical. A toxicity difference due to different impurity profiles is not likely to occur.

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]


Justification for the use of 3,5,5-trimethylhexanoic acid as source chemical:

- 3,5,5-trimethylhexanoic acid is the presumed metabolite of 6-(isononanoylamino)hexanoic acid. It is also the presumed metabolite of 6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol.
This view is based on the results of the metabolites investigation in degradation samples obtained in a Zahn-Wellens test. The β-oxidation at the terminal carboxylic acid moiety in combination with hydrolysis at amide bond could be assumed as the degradation pathway leading to 3,5,5-trimethylhexanoic acid as a stable metabolite.
Also the hydrolysis at the amide moiety is thinkable. The hydrolysis products would be then 3,5,5-trimethylhexanoic acid and 6-aminocaproic acid. The latter compound is a drug known as amicar and is expected to be far more rapidly eliminated than 3,5,5-trimethylhexanoic acid. A significant toxicity attribution of 6-aminocaproic acid cannot be derived.
One literature article was found, describing further biotransformation of 3,5,5-trimethylhexanoic acid in rat: gamma-lactone of 3,5,5-trimethylhexanoic acid is formed, which may be the ultimate toxicant for the alpha-2u-globulin accumulation in kidneys.
As mentioned above no significant toxicity contribution of 2,2`,2``-nitrilotriethanol is expected.

-The findings in the 28-day oral toxicity study on the 3,5,5-trimethylhexanoic acid are comparable to those found in the repeated dose toxicity studies of the target chemical and 6-(isononanoylamino)hexanoic acid:
- liver and kidney enlargement
- peroxisome proliferation in the liver
- The alpha-2u-globulin accumulation in kidneys.

4. DATA MATRIX

Data matrix and other information see the attached read-across justification in chapter 13

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

no histological examinations

Principles of method if other than guideline:

one generation range-finding study similar to guideline OECD 421 and performed according to GLP guidelines. No histological examination of the reproductive or other organs were performed.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,5,5-trimethylhexanoic acid

EC Number:

221-975-0

EC Name:

3,5,5-trimethylhexanoic acid

Cas Number:

3302-10-1

Molecular formula:

C9H18O2

IUPAC Name:

3,5,5-trimethylhexanoic acid

Test animals

Species:

rat

Strain:

other: Crl:CDBR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Inc., males: Raleigh, NC, USA, females: Stone Ridge, NY, USA
- Age at study initiation: (P) approx. 7 wks
- Weight at study initiation: (P) Males: 221-266 g; Females: 168-201 g
- Housing: individual, except the mating and post partum periods
- Diet: PMI Certified Rodent Diet Meal 5002 ad libitum
- Water: ad libitum
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24,4
- Humidity (%): 40-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: undiluted test substance used

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly (first 5 weeks), twice weekly (therafter)
- Mixing appropriate amounts with (Type of food): basal diet
- Storage temperature of food: room temperature

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug and/ or sperm in vaginal smear, referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All meal samples were within 10% of nominal concentration; stable for at least 4 days at 0.06% concentration, for at least 15 days at higher concentrations, details given in Appendix W

Duration of treatment / exposure:

P1 males and females were both exposed for at least 10 weeks proir to mating, through the mating period. Males were further exposed until their sacrifice, females throughout gestation and lactation until day 28 post partum. F1 pups were exposed from day 28 post partum until their sacrifice (postnatal day 42 (females) to 49 (males)).

Frequency of treatment:

daily, via feed

Doses / concentrations

Remarks:

Doses / Concentrations:
0.06, 0.12, 0.25 and 0.5%
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cageside observations were performed daily on all P1 adults and after weaning for all F1 offspring, except the days clinical observations were performed.

VIABILITY: Yes
-Tiime schedule: All animals were examined for viability at least twice daily Monday through Friday, and at least once daily on weekends and holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A clinical examination was given to each male prior to P1 selection, on the first day of dosing, and at least weekly thereafter until euthanized. Females received a clinical examination prior to P1 selection, on the first day of dosing, and at least weekly thereafter until confirmation of mating, then on GD 0, 7, 14, and 21, and on PPD 0, 4, 7, 14 and 21.


BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded for all females pretest, at initiation of dosing (Day 0), on Days 4 and 7, and weekly until confirmation of mating or the end of the mating period. Body weights were recorded for confirmed-mated females on GDs 0, 7, 14, and 21, on PPDs 0, 4, 7, 14, and 21 and weekly after Postpartum Day 21. After the mating period, body weights were recorded weekly for females not confirmed mated until they were sacrificed. Confirmed-mated females which did not deliver by GD 26 were weighed weekly after GD 26 until sacrificed. Body weights also were recorded on the day of sacrifice for all females.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured on Days 1, 2, 3, and 7, and then concurrently with body weight after Day 7, except during the mating period and on GD 0 and PPD 0 for the females when food consumption was not measured.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


OTHER:
- signs of parturition were examined at least twice daily beginning on gestational day (GD) 21. The duration of gestation was calculated and any difficulties occurring at parturition were noted. The date of parturition was recorded as the dam's Postpartum Day 0 (PPD 0).

POSTNATAL EXAMINATION: Yes
Each morning and afternoon during the postnatal period, the litters were checked for dead offspring and unusual conditions, and the dams were examined for viability, nesting, and nursing behavior.
Dead pups were removed from the litter immediately after their discovery. If intact, dead pups were examined externally and internally for anomalies. Dead pups discovered on PND 0 also were examined internally to determine whether they were stillborn.
On PND 0, 1, 4, 7, 14, 21, and 28 the offspring were counted, sexed, and each live pup was weighed. Pups were counted and examined externally on a daily basis during the postnatal period. All animals were weighed on PND 35, 42, and 49 (males only were weighed on PND Day 49).
On PND 4, after counting, weighing, and examining the pups, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter. Partial adjustment (e.g., 5 males and 3 females) was permitted whenever there were not enough pups to obtain 4 per sex per litter. Litters of eight pups or less were not adjusted.
Culled pups were sacrificed. Culled pups that appeared normal received only an external examination and tissues were not saved. Culled pups that appeared abnormal were subjected to a visceral examination.
The pups from each litter were examined daily for pinna detachment (starting PND 1), hair growth (starting PND 3), righting reflex (starting PND 3), incisor eruption (starting PND 7), and eye opening (starting PND 11). The examinations continued for an individual landmark until the criterion for that landmark was attained. Additionally, beginning on PND 29, all surviving female offspring were examined daily for vaginal opening. Beginning on PND 35 all surviving male offspring were examined daily for preputial separation. The examinations continued until all animals reached criteria.

Oestrous cyclicity (parental animals):

Vaginal smears were performed on each female on their day of sacrifice to determine its stage in the estrous cycle. The stage of the estrous cycle was recorded, but not used for estrous cycle calculations.

Sperm parameters (parental animals):

Samples of sperm from the left distal cauda epididymis (or proximal vas deferens) were collected at necropsy and evaluated for the percentage of progressively motile sperm and sperm morphology. Also, the entire left cauda epididymis was minced in saline to enumerate the total number of sperm (cauda reserves).

Litter observations:

Each morning and afternoon during the postnatal period, the litters were checked for dead offspring and unusual conditions, and the dams were examined for viability, nesting, and nursing behavior.
Dead pups were removed from the litter immediately after their discovery. If intact, dead pups were examined externally and internally for anomalies. Dead pups discovered on PND 0 also were examined internally to determine whether they were stillborn.
On PND 0, 1, 4, 7, 14, 21, and 28 the offspring were counted, sexed, and each live pup was weighed. Pups were counted and examined externally on a daily basis during the postnatal period. All animals were weighed on PND 35, 42, and 49 (males only were weighed on PND Day 49).
On PND 4, after counting, weighing, and examining the pups, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter. Partial adjustment (e.g., 5 males and 3 females) was permitted whenever there were not enough pups to obtain 4 per sex per litter. Litters of eight pups or less were not adjusted.
Culled pups were sacrificed. Culled pups that appeared normal received only an external examination and tissues were not saved. Culled pups that appeared abnormal were subjected to a visceral examination.
The pups from each litter were examined daily for pinna detachment (starting PND 1), hair growth (starting PND 3), righting reflex (starting PND 3), incisor eruption (starting PND 7), and eye opening (starting PND 11). The examinations continued for an individual landmark until the criterion for that landmark was attained. Additionally, beginning on PND 29, all surviving female offspring were examined daily for vaginal opening. Beginning on PND 35 all surviving male offspring were examined daily for preputial separation. The examinations continued until all animals reached criteria.

Postmortem examinations (parental animals):

Gross necropsies were performed on all adult animals that were found dead. Body weight was recorded on the day of necropsy. The uterus of each female used for mating, but failing to deliver, was examined grossly for evidence of implantations and these data were recorded.
A gross necropsy was performed on all adult animals surviving to termination. Body weights were recorded on the day of necropsy. The uterus of each female was examined grossly for evidence of implantation and the number of implantation sites was recorded.

The following tissues and organs of all males surviving to termination and all females were weighed prior to fixation:
ovaries (individual) uterus
testes (individual) prostate
liver
right epididymis (total and cauda)
seminal vesicles (with coagulating glands and their fluids)

The following organs and tissues of all adults were preserved in 10% neutral buffered formalin:
coagulating gland right epididymis
seminal vesicles prostate
testes* uterus
liver ovaries
*:The right testis was preserved in Bouin's solution. The right testis remained in Bouin's solution for approximately 24 hours. The right testis was then rinsed with tap water and stored in 70 percent Ethyl Alcohol. The left testis was frozen for enumeration of homogenization resistant spermatids.

Abnormal tissues were preserved in 10% neutral buffered formalin at the discretion
of the Study Director or designee for possible future microscopic examination.

Postmortem examinations (offspring):

Intact dead pups or pups sacrificed in moribund condition on PND 0 were examined by fresh visceral dissection. Dead pups and pups sacrificed as moribund after PND 0 were examined externally for anomalies and internally for gross visceral abnormalities. Culled pups (PND 4) with external abnormalities were subjected to a visceral examination at the discretion of the Study Director or his designee.

Statistics:

Group means and standard deviations were calculated.

Reproductive indices:

mean male fertility and mating indices and female fertility, fecundity and gestational indices

Offspring viability indices:

offspring survival

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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:

not examined

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Significant increases in body weight gain in males of the 0.12 and 0.5% dose groups were considered incidental and unrelated to treatment with the test material (absence of a clear consistent response over the test period).
Statistically significantly decreased mean body weights when compared with controls were observed in the 0.5% females on Gestation Day (GD) 7 (9%) and on GD 21 (11%) and on Postpartum Days (PPD) 4, 7, and 14 (17%, 12%, and 10%, respectively). Also, there was a corresponding statistically significant decrease in mean body weight change for the 0.5% group females at the PPD 0-4 interval (209%). There also was a statistically significantly decreased mean body weight compared with controls in the 0.25% females on PPD 4 (10%).
There also were statistically significant increases in mean body weight change in the 0.25 and 0.5% group females at the PPD 4/7 (470% and 567%, respectively), PPD 14/21 (154% and 235%, respectively), and in the 0.25% females on PPD 0/21 intervals (112%).
There were statistically significant decreases in mean food consumption for the 0.5% group males at Days 1 (2 1%) and 2 (2 1%). There were statistically significant decreases in mean food consumption in the 0.25% group females at Day 1 (30%), Day 3 (18%), and Weeks 1 (13%), 2 (11%), 3 (21%), 4 (13%), and 6 (12%). There also were statistically significant decreases in mean food consumption for the 0.5% group females at Day 1 (47%) and Weeks 1 (15%), 2 (13%), 4 (11%), and 6 (10%). These decreases were considered to be due to reduced palatability of the diet mixtures.
There were no statistically significant differences in food consumption during the gestation period. However, statistically significant decreases in food consumption were noted in the 0.5% group females during the PPD 0/4 (46%), 7/14 (15%), and entire postpartum period (PPD 0-28) (17%).


ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no statistically significant changes in the mean absolute or mean relative organ weights for the reproductive organs weighed during the study. There were statistically significant increases in the mean absolute and mean relative liver weights of the 0.5% males (15% and 21%, respectively) and 0.5% females (21% and 24%, respectively) and the 0.25% females mean absolute and relative liver weights (15% and 14%, respectively). The significance of the increases in the liver weights could not be determined because histopathology was not performed on the tissues.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
There was a statistically significant decrease in the mean percent live offspring (6%) and the corresponding increase in the mean percent dead offspring (700%) in the 0.5% dose group.

Effect levels (P0)

1

Dose descriptor:

LOAEL

Effect level:

165 - 500 mg/kg bw/day

Sex:

female

Basis for effect level:

other: decreases in body weight, increased liver weight; 0.25% in diet, calculated by the authors on the basis of food consumption and body weights

2

Dose descriptor:

NOAEL

Effect level:

79 - 228 mg/kg bw/day

Sex:

female

Basis for effect level:

other: 0.12% in diet, calculated by the authors on the basis of food consumption and body weights

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
There were statistically significant decreases in 0.5% group offspring compared with the control offspring for the live birth index (6.0%), the Day 1 survival index (14%), and the Day 4 survival index (2 1%).

BODY WEIGHT (OFFSPRING)
There were statistically significant decreases in mean offspring body weights in the 0.25% group males and females at PND 0, 1, 4, 7, 35 and 42. There also were statistically significant decreases in mean offspring body weights in the 0.25% group males at PND14, 28, and 49.

SEXUAL MATURATION (OFFSPRING)
There was a statistically significant advance for preputial separation for the 0.06% group males (1.2 days) when compared with the controls. Due to the small size of this advance and the absence of a dose response, this difference was not considered biologically significant. There also was a statistically significant retardation of preputial separation for the 0.5% group males (2.1 days) compared with the control male offspring. In the females, the 0.5% group exhibited a statistically significant retardation (1.8 days) for vaginal patency compared with controls. These two findings are not biologically significant but rather reflect the normal maturation of these animals with the delays due to somewhat smaller body weights.

OTHER FINDINGS (OFFSPRING). DEVELOPMENTAL LANDMARKS
There were statistically significant retardations in the male eye opening for the 0.5% group (0.7 days later), and the male and female pinna detachment in the 0.5% group (1.0 day later each). These findings are not biologically significant but rather reflect the normal maturation of these animals with the delays due to somewhat smaller body weights.

Effect levels (F1)

1

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

0.25 other: % in diet

Sex:

male/female

Basis for effect level:

other: reduced fetal weights, no calculation of body doses stated

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

0.12 other: % in diet

Sex:

male/female

Basis for effect level:

other: no calculation of body doses stated

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

BODY DOSES BASED ON FOOD CONSUMPTION AND BODY WEIGHTS

	DOSE (%)	WEEK 1 (mg/kg/day)	WEEKS 2-9 (mg/kg/day)	WEEK 10 (mg/kg/day)	Gestation	Postpartum
M A L E S	0.06	59	33-51	32	Not Applicable	Not Applicable
	0.12	117	66-101	66
	0.25	247	147-215	145
	0.5	477	289-435	290
F E M A L E S	0.06	57	40-56	40	37-42	57-126
	0.12	116	79-112	81	79-86	109-228
	0.25	231	168-224	167	165-185	241-500
	0.5	450	347-453	347	336-382	311-970

MEAN OFFSPRING BODY WEIGHT - F1 (PREWEANING)

}Group	MALE PND 0	MALE PND 1	MALE PND 4	MALE PND 7	MALE PND 14	MALE PND 21	MALE PND 28
0%	6.71	7.09	9.54	15.58	32.32	51.18	91.53
0.06%	6.82	7.31	10.13	16.70	34.12	53.06	93.88
0.12%	6.39	6.85	9.01	14.44	30.72	48.88	90.23
0.25%	5.76**h	5.95**h	7.98*h	12.55**h	28.64*h	46.23	81.65**
0.5%	5.66**h	5.73**h	7.67**h	11.07**h	23.31**h	39.18**h	72.63**
Historical Control	6.35-7.02	6.68-7.49	8.53-11.43	13.64-18.74	28.81-37.09	44.89-62.34	98.34
Group	FEMALE PND 0	FEMALE PND 1	FEMALE PND 4	FEMALE PND 7	FEMALE PND 14	FEMALE PND 21	FEMALE PND 28
0%	6.36	6.89	9.33	14.72	30.50	47.77	82.46
0.06%	6.51	7.00	9.67	15.35	31.89	49.18	83.84
0.12%	5.92h	6.40	8.78	13.81	28.79	46.18	81.83
0.25%	5.46**h	5.65**h	7.83**h	12.77*h	28.89	45.33	77.09
0.5%	5.65**h	5.65**h	7.55**h	11.10**h	23.47**h	38.59**h	69.34**
Historical Control	5.96-6.74	6.30-7.16	8.32-11.05	13.33-17.69	27.22-35.89	42.39-61.19	90.68

MEAN OFFSPRING BODY WEIGHT - F1 (POSTWEANING)

{ }Group	MALE PND 35	MALE PND 42	MALE PND 49
0%	147.7	205.8	266.3
0.06%	151.0	211.4	272.4
0.12%	144.3	205.1	266.3
0.25%	128.4**	183.4**	242.1**
0.5%	119.8**	170.9**	227.6**
Group	FEMALE PND 35	FEMALE PND 42	FEMALE PND 49
0%	124.8	158.3	NA
0.06%	126.5	161.4	NA
0.12%	123.1	157.2	NA
0.25%	114.9**	148.6*	NA
0.5%	109.3**	140.9**	NA

NOTE: All weights are in grams

* Mean significantly different from control mean (p0.05)

** Mean significantly different from control mean (p0.01)

NA not applicable

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study administration of the test substance resulted in evidence of maternal toxicity and findings in offspring that may have been secondary to maternal toxicity. Maternal toxicity was demonstrated by decreased body weight and food consumption during the gestation and postpartum periods, and increased absolute and relative liver weights in the 0.25% and 0.5% groups (79-228 and 165-500 mg/kg bw and day). Paternal effects were limited to increased absolute and relative liver weights in the 0.5% group. The differences between male and female adults in this study may be due to the somewhat higher dose rates observed in the females, especially during the postpartum period. Coincident with the maternal effects, offspring body weights were decreased in both sexes in the 0.25% and 0.5% groups, and offspring survival was decreased in the 0.5% dose group. There were also some small delays in developmental landmarks in the high dose offspring. These delays are likely secondary to the decreased offspring growth in this group.
There was no evidence of adverse effects on mating behavior, fertility, sperm parameters, or reproductive organ weights in the parental animals. Thus, the test material does not effect fertility at the dose levels tested. There were also no malformations observed in the offspring in this study. There were indications of offspring toxicity that may be related to maternal toxicity in the two highest doses. The apparent NOAEL for both maternal and offspring effects in this study is the 0.12% level.

Executive summary:

In this one-generation range-finding study (performed similar to OECD guideline 415) the test substance was administered to male and female rats in the diet (10 per sex and dose; 0.06, 0.12, 0.25 and 0.5% in diet) for at least ten weeks prior to mating and during the mating period. The dams were exposed during the gestation and postpartum periods, until weaning of the offspring on Postpartum Day (PPD) 28. The parental male animals were sacrificed at the end of the mating period, the females and the offspring after weaning.

Effects in the P-generation:

There were no treatment-related deaths, clinical signs or effects on male or female reproductive organ weights or reproductive parameters. Statistically significant decreases were observed in food consumption of the 0.5% group females at various postnatal timepoints. The body weights of the parental generation were reduced in females of the 0.25% group at PPD 4 as well as in 0.5% females at Gestation Days (GD) 7 and 21 and at PPD 4, 7, and 14. Liver weights were increased in males at 0.5%, in females at 0.25% and above.

Effects in offspring:

Reduced Live Birth Index and survival indices were observed in the 0.5% group. Postnatal offspring body weights of both sexes of the 0.25% and 0.5% groups were reduced compared with the controls.

No treatment-related clinical signs were noted for the offspring. A postnatal developmental delay was obvious in the 0.5% dose group offspring in form of delayed onset of eye opening in male offspring, pinna detachment in both sexes, retardation of preputial separation and vaginal patency. These delays were considered to be treatment-related and secondary to reduced offspring body weight.

No effects were observed with respect to fertility, thus the NOAEL for reproductive toxicity was 0.5% (highest tested dose, 289-477 mg/kg bw/day in males, 336-970 mg/kg bw/d in females for P generation). The LOAEL for systemic parental (female) and offspring toxicity was 0.25% (165 -500 mg/kg/bw/day for P generation), the NOAEL was 0.12% (79 -228 mg/kg bw/day for P generation)(Exxon, 1998).

This study, performed similar to OECD guideline 415, was judged to be reliable (RL2) and selected as key study.