Tall oil, compd. with diethanolamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Study period:

8 April 2002 to 15 July 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability of 2 given since the data is based on read across, not the target substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil

EC Number:

232-304-6

EC Name:

Tall oil

Cas Number:

8002-26-4

IUPAC Name:

Tall Oil

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD (SD) IGS BR (outbred albino).
- Age at arrival: Around 6 weeks.
- Weight at arrival: 180 to 190 g for the males; 113 to 161 g for the females.
- Fasting period before study: No.
- Housing: The animals were housed 2 per cage initially, in polypropylene cages (42 x 27 x 20 cm) with stainless steel grid bottoms and mesh tops. A stainless steel food hopper and a polypropylene or polycarbonate water bottle were provided for each cage. Excreta were collected on a tray lined with absorbent paper suspended beneath each cage. Male and female cages were racked separately.
A few days prior to pairing for mating, males were transferred to individual grid-bottomed cages (59 x 38.5 x 20 cm) of similar design. Mated females were transferred to individual (42 x 27 x 20 cm) solid bottomed cages. Sterilised white wood shavings were provided as bedding and white paper tissue as nesting material where appropriate. Wooden chewsticks were provided for environmental enrichment.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): The animals had access to domestic mains water ad libitum via water bottles.
- Acclimation period: 12 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2 °C.
- Humidity (%): 50 ± 15 %.
- Air changes (per hr): Minimum of 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Automatic control of the 12 hour light cycle; light from 0700 to 1900 hours.

IN-LIFE DATES: From: 3 April 2002 To: 27 May 2002

Administration / exposure

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
An appropriate quantity of the test material was dissolved in a suitable volume of acetone. This solution was added to a suitable quantity of untreated diet and mixed for around one hour with fan assisted venting to form a dose premix. A control premix was prepared using the same proportion of acetone and untreated diet.
The diets for the intermediate and high dose groups were prepared by dilution of the dose premix with untreated diet to give the desired concentrations. The low dose diet was prepared by dilution of the high dose diet with untreated diet. The diet premixes were then placed on a Winkworth mixer for about 20 minutes.
The control diet was prepared by dilution of the control premix with untreated diet such that the diet contained the same proportion of premix as the high dose diet.

Details on mating procedure:

MATING PROCEDURE
Pairing was on a one male to one female basis, within the same treatment group. Each female was transferred to the cage of an appropriate co-group male near the end of the working day, and remained there until mating was detected.
Vaginal lavages were taken early each morning commencing on the day of pairing, until mating was detected, and the day of observation of a copulatory plug in situ and/or sperm in the lavage was designated day 0 of gestation.
Each female remained with its first designated male for a maximum of 7 consecutive nights.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DIET FORMULATIONS
The analytical method was validated in a separate study at the testing facility.
Diet formulations were sampled twice during the study (weeks 1 and 4). Triplicate samples of each formulation, including control, were taken immediately after preparation. On both occasions, the analysed concentrations were found to be within ± 10 % of the nominal concentration, indicating acceptable accuracy of formulation. A low coefficient of variation (3.4 % or less) was indicative of satisfactory homogeneity.

Duration of treatment / exposure:

The males were dosed for at least 4 weeks overall, starting from 2 weeks prior to mating until termination.
The females were dosed for 2 weeks prior to mating, then through mating until termination after day 4 of lactation.

Frequency of treatment:

Continuous in the diet.

Details on study schedule:

- Age at mating of the mated animals in the study: approximately 10 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1000, 5000 and 20 000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of existing toxicological data, including a one week range finding study in the rat.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All the animals were examined for reaction to treatment on each day. The nature, onset, duration and intensity of any signs were recorded. In addition, all the animals were checked for viability early in the morning and again as late as possible on each day.

BODY WEIGHT: Yes
- Time schedule for examinations: Male weights were recorded once during the week prior to the commencement of dosing and once weekly thereafter until termination.
Female weights were recorded once during the week prior to the commencement of dosing, and weekly thereafter until the start of the mating period, and then on day 0 of gestation (the day of detection of a positive mating sign) followed by days 7, 14 and 20 of gestation, and then days 1 and 4 of lactation (where day 0 = the day of parturition).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
For all animals, the weight of food consumed by each cage was recorded once weekly, beginning during the week prior to the commencement of dosing, until pairing for mating. All animals were fed ad libitum during mating, but following completion of mating weekly consumption was recommenced for males, until termination.
For mated females, the amount of food consumed was recorded over days 0 to 7, 7 to 14 and 14 to 20 of gestation, and days 0 to 4 of lactation.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND COAGULATION: Yes
- Time schedule for collection of blood: The blood samples were collected from the lateral tail vein after careful cleaning, during week 5 of dosing for males and on day 6 of lactation for females. A sample of 0.5 mL was transferred into tubes containing EDTA for haematology investigations. A further 0.45 mL was taken into tubes containing 0.05 mL 3.8 % (w/v) trisodium citrate for assessment of coagulation; the final sample volume was to be as close as possible to 0.5 mL to give a final concentration of 0.38 % (blood to citrate ratio of 9:1).
- Animals fasted: No
- How many animals: Samples were obtained from 5 males and 5 females from each dose group. For males the first 5 animals in each group were tested. For females the first 5 animals to have reared their litter to day 6 of lactation were tested.
- Parameters examined for haematology: Haemoglobin, Red Blood Cell Count, Haematocrit, White Blood Cell Count, Mean Cell Volume, Mean Cell Haemoglobin, Mean Cell Haemoglobin Concentration, Platelets, Differential White Blood Cell Count: Neutrophils; Lymphocytes; Monocytes; Eosinophils; Basophils; Large Unclassified Cells.
- Parameters examined for coagulation: Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The blood samples were collected from the lateral tail vein after careful cleaning, during week 5 of dosing for males and on day 6 of lactation for females. At least 1 mL was taken into lithium heparin tubes to be used for clinical chemistry investigations.
- Animals fasted: No
- How many animals: Samples were obtained from 5 males and 5 females from each dose group. For males the first 5 animals in each group were tested. For females the first 5 animals to have reared their litter to day 6 of lactation were tested.
- Parameters examined: Urea, Glucose, Aspartate Aminotransferase, Alanine Aminotransferase, Sodium, Potassium, Chloride, Total Protein, Albumin, A:G Ratio; Creatinine, Calcium, Phosphate, Total Bilirubin, Cholesterol, Alkaline Phosphatase.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight and epididymis weight.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies and physical abnormalities.


GROSS EXAMINATION OF DEAD PUPS: Yes, for external abnormalities.

Postmortem examinations (parental animals):

GROSS PATHOLOGY
All animals were sacrificed by exposure to carbon dioxide, weighed then exsanguinated by severance of major blood vessels. All adults were subject to a detailed necropsy under the guidance of a veterinary pathologist. The necropsy consisted of an external and internal examination. All gross lesions were recorded in terms of location, size, shape, colour, consistency and number. The pups were sacrificed at the same time as their mother.

HISTOPATHOLOGY
Histological examination was conducted on control and high dose animals only, the same animals that were used for haematology, coagulation and clinical chemistry evaluations.
The following organs were removed from all animals and were weighed and preserved as appropriate: Abnormal Tissue (included local lymph nodes to masses), Adrenals, Aorta, Brain (Forebrain, mid-brain, cerebellum and pons), Ears, Epididymides, Eyes, Gastro-Intestinal Tract (Stomach; Duodenum; Jejunum; Ileum; Caecum; Colon; Rectum), Heart, Kidneys, Liver, Lung, Marrow Smear (femur), Mesenteric Lymph Node, Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Sciatic Nerve, Seminal Vesicles, Skin and Mammary Gland, Spinal Cord (Cervical, subthoracic and lumbar regions), Spleen, Sternum, Submandibular Lymph Node, Submaxillary Salivary Gland, Testes, Thymus, Thyroid with Parathyroid x 2, Trachea, Urinary Bladder, Uterus.

PROCESSING OF FIXED TISSUES
Tissues were trimmed to a maximum thickness of 3 mm for processing. Parenchymal organs were trimmed to allow the largest surface area possible for examination.
Hollow organs were trimmed and blocked to allow preparation of a cross section from mucosa to serosa.
Sections were cut at 4 to 6 µm thickness and stained with haematoxylin and eosin. An additional section from each testis was stained with Periodic Acid Schiff and haematoxylin.

Postmortem examinations (offspring):

SACRIFICE
The pups were sacrificed by injection of sodium pentobarbitone.

GROSS NECROPSY
The pups were examined for visible external abnormalities.

Statistics:

- Bodyweight and food consumption (prior to mating for females), haematology and clinical chemistry data were statistically analysed for homogeneity of variance using the ‘F-max’ test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons made via Student’s t-test using Fisher’s F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous then Kruskal-Wallis ANOVA was used.
- Organ weights were also analysed as above and by analysis of covariance (ANCOVA) using terminal kill bodyweight as covariate.
- Histology incidence data were analysed using Fisher’s Exact Probability Test.
- The following pairwise comparisons were performed against the control group: control vs low dose; control vs intermediate dose; control vs high dose.

All statistical tests were two-sided and performed at the 5 % significance level.

Reproductive indices:

The following indices of fertility were evaluated for each group.

- Fertility Index (female) = Number pregnant / Number paired

- Fertility Index (male) = Number siring a Litter / Number paired

- Gestation Index = Number bearing live pups / Number pregnant

Offspring viability indices:

The following were evaluated for each group.

- Birth Index = Total number of pups born (live and dead) / Number of implantation scars

- Live Birth Index = Number of pups live on day 0 of lactation / Total number born (live and dead)

- Viability Index = Number of pups live on day 4 of lactation / Number live on day 0

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

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: see below

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

BODYWEIGHTS
At 20 000 ppm there was a transient decrease in weight gain in both sexes. In males, decreased weight gain was most notable over the first week, although absolute weights were significantly lower over the first 3 weeks of treatment. In females, there was a notable decrease throughout the pre-mating phase. The resulting deficit in body weight was never regained in either sex. In pregnant females reduced weight gain was evident over days 7 to 20 of gestation, compared to the control animals.
There were no obvious effects of treatment at 5000 or 1000 ppm.

FOOD CONSUMPTION AND ACHIEVED DIETARY INTAKE
At 20 000 ppm food consumption in males was reduced for the first 2 weeks of treatment (attaining significance during week 1) and in week 4 (not recorded week 3 as paired for mating). In females, food consumption was significantly decreased during the pre-mating period. Consumption was also reduced during the first half of the gestation period, compared to the control animals.
There were no obvious effects of treatment at 5000 or 1000 ppm.
The achieved intake in the first week of treatment for males and females at 20 000 ppm was lower than the second week. For males and females at the low and intermediate dose levels, intake was higher than in the following weeks (as expected). At other times, the achieved intake was essentially proportional to the diet concentrations.

HAEMATOLOGY
At 20 000 ppm there was a non-significant decrease in white blood cells in females. Any other intergroup differences were not considered to reflect an effect of treatment.

CLINICAL CHEMISTRY
Alkaline phosphatase levels were significantly increased in females at 5000 and 20 000 ppm, and in males at 20 000 ppm. In males, there was a non-significant increase in levels at 5000 ppm and in females at 1000 ppm there was an equivocal increase, but given the small group size it was considered that the difference was too small to reflect an effect of treatment.
Total bilirubin was increased in both sexes at 20 000 ppm.
In addition, at 20 000 ppm, cholesterol levels were increased in males; albumin (and consequently total protein) were reduced in females.

ORGAN WEIGHTS
In males, at 20 000 ppm there was a decrease in bodyweights, with liver weights being essentially similar to controls. At 5000 and 1000 ppm liver weight was slightly greater than controls. Following covariance analysis, there was a dose related increase in liver weights, with the increases at 5000 and 20 000 ppm attaining statistical significance.
In females, slight non-significant increases in liver weights following covariance analysis at 5000 and 20 000 ppm were too small to attribute to treatment.
In males at 20 000 ppm, spleen weight was notably increased following variance and covariance analysis. Adrenal gland and thymus weights were slightly but significantly decreased. Following covariance analysis, adrenal gland weight was still significantly decreased, but for the thymus there was no significant difference from controls.
In females, ovary, adrenal gland and kidney weights were significantly reduced at 5000 and 20 000 ppm, with pituitary gland weight reduced at 20 000 ppm. Following covariance analysis, kidney and pituitary gland weights were essentially similar to controls, but a decrease in ovary weight at 20 000 ppm and adrenal gland weight at 5000 and 20 000 ppm was still evident, but not significant.

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At 20 000 ppm, decreased weight gain and food consumption and changes in liver function were seen in both sexes. At 5000 ppm increased alkaline phosphatase levels were seen in both sexes.

2

Dose descriptor:

NOEL

Effect level:

5 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There was a marginal decrease in implant sites at 20 000 ppm with a corresponding decrease in the mean total number of pups born compared to other dose groups.

Remarks on result:

other: Generation: Reproductive Parameters (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

REPRODUCTIVE PARAMETERS
- Mating Performance and Duration of Gestation
Mating performance was not affected by treatment.
There were no obvious effects on the duration of gestation at any of the dose levels applied.

- Litter Size and Survival
At 20 000 ppm, the mean number of implant sites per pregnancy was marginally decreased and hence the mean total number of pups born was lower than that of all other dose groups. However, due to the very slight differences compared to the control group, there is some doubt as to the reproducibility of this finding.
Litter survival, as indicated by the birth index and viability index, was similar in all groups.

- Litter and Pup Weights
At 20 000 ppm mean litter weights were slightly reduced compared to the controls, reflecting the decrease in litter size.

- Abnormalities among Pups
There were no obvious external abnormalities noted in the pups at any of the dose levels applied.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1 Parental Pre-mating Mean Group Bodyweights (g)

Dose Level (ppm)		Males							Females
		Pre-trial (days)		Treatment Period (days)				Body-weight Gain day 0 to 28	Pre-trial (days)		Treatment Period (days)		Body-weight Gain day 0 to 14
		-7	0	7	14	21	28		-7	0	7	14
0	Number Mean SD	10 249 10	10 320 17	10 374 26	10 414 34	10 447 37	10 468 45	10 149 30	10 165 8	10 194 9	10 217 13	10 237 16	10 43 9
1000	Number Mean SD	10 248 4	10 313 9	10 369 16	10 408 17	10 437 19	10 460 23	10 147 16	10 166 12	10 199 15	10 221 18	10 240 22	10 41 12
5000	Number Mean SD	10 245 6	10 316 11	10 373 16	10 415 20	10 444 26	10 471 28	10 154 20	10 167 6	10 197 8	10 219 10	10 237 10	10 40 4
20 000	Number Mean SD	10 245 8	10 313 10	10 351 13**	10 386 18*	10 413 22**	10 434 29	10 121 24*	10 156 7	10 188 9	10 200 9**	10 214 15**	10 25 12**

Significantly different from the control: *p<0.05; **p<0.01; ***p<0.001

SD = Standard Deviation

 

Table 2 Parental Bodyweights (Females) During Gestation and Lactation Mean Group Values (g)

Dose Level (ppm)	Day of Gestation				Weight Gain Days 0 to 20 of Gestation	Percentage of Control	Day of Lactation
	0	7	14	20			1	4
0	245	286	329	410	165	-	298	319
1000	247	285	320	407	160	97	286	313
5000	240	275	317	388	148	90	273	300
20 000	218	256	288	345	127	77	261	281

 

Table 3 Parental Food Consumption Group Mean Values (g/animal/day)

Dose Level (ppm)		Males				Females
		Pre-trial (day)	Treatment Period (days)			Pre-trial (day)	Treatment Period (days)
		0	7	0	28	0	7	14
0	Number Mean SD	5 29.8 1.8	5 31.5 2.2	5 35.5 3.9	5 32.0 2.8	5 21.2 0.8	5 21.1 1.0	5 29.2 1.3
1000	Number Mean SD	5 27.9 1.1	5 30.9 2.0	5 34.1 1.9	5 31.5 1.5	5 20.5 0.7	5 21.7 1.2	5 22.6 1.1
5000	Number Mean SD	5 30.1 1.3	5 32.6 1.6	5 34.9 1.3	5 32.4 2.5	5 19.7 1.9	5 20.5 2.0	5 21.6 1.5
20 000	Number Mean SD	5 29.6 1.1	5 27.2 2.2**	5 32.1 2.4	5 29.9 1.9	5 21.0 1.7	5 17.2 1.3***	5 20.3 1.5**

Significantly different from the control: *p<0.05; **p<0.01; ***p<0.001

SD = Standard Deviation

 

Table 4 Parental Food Consumption (Females) During Gestation and Lactation Mean Group Values (g/animal/day)

Dose Level (ppm)	Day of Gestation			Day of Lactation
	0 to 7	7 to 14	14 to 20	0 to 4
0	26.9	29.9	31.8	33.9
1000	26.5	30.0	32.8	31.9
5000	26.2	27.9	32.6	31.6
20 000	23.9	26.7	30.1	30.1

  

Table 5 Selected Parental Absolute Organ Weights Group Mean Values

		Male				Female
		Dose Level (ppm)				Dose Level (ppm)
		0	1000	5000	20 000	0	1000	5000	20 000
Body Weight (g)	Number Mean SD	10 470 48	10 461 24	10 469 30	10 432 28	10 329 22	10 314 17	10 306 14*	8 287 23***
Adrenal Glands (g)	Number Mean SD	10 0.0758 0.0106	10 0.0803 0.0097	10 0.0675 0.0154	10 0.0631 0.0070*	10 0.0944 0.0118	10 0.0849 0.0068	10 0.0784 0.0122**	8 0.0732 0.0109***
Ovaries (g)	Number Mean SD	-	-	-	-	10 0.117 0.018	10 0.106 0.010	10 0.103 0.011*	8 0.093 0.014***
Kidneys (g)	Number Mean SD	10 3.86 0.52	10 3.87 0.38	10 4.10 0.29	10 3.86 0.44	10 2.71 0.17	10 2.56 0.18	10 2.44 0.17**	8 2.38 0.26**
Liver (g)	Number Mean SD	10 18.50 2.57	10 19.05 2.72	10 20.04 2.56	10 18.76 1.42	10 17.65 1.79	10 16.34 1.71	10 17.58 1.32	8 16.68 1.75
Pituitary Gland (g)	Number Mean SD	10 0.013 0.002	10 0.012 0.003	10 0.013 0.001	10 0.011 0.002	10 0.015 0.003	10 0.016 0.003	10 0.015 0.002	8 0.012 0.002**
Spleen (g)	Number Mean SD	10 0.87 0.11	10 0.84 0.10	10 0.87 0.12	10 0.99 0.14*	10 0.65 0.11	10 0.64 0.08	10 0.67 0.10	8 0.66 0.08
Thymus (g)	Number Mean SD	10 0.507 0.141	10 0.419 0.111	10 0.450 0.102	10 0.350 0.054**	10 0.239 0.085	10 0.240 0.076	10 0.193 0.078	8 0.192 0.072

Table 6 Group Mean Duration of Gestation and Overall Litter Performance Values

Dose Level (ppm)	Number Pregnant	Duration of Gestation (days)				No. of Females Producing a Live Litter	Gestation Index as a %	Mean No. Implant Sites Per Pregnancy (± SD)	Mean Total No. Pups Born	Mean No. of Live Pups Per Litter (± SD)
		21	22	23	Mean					Day 0 Lactation	Day 1 Lactation	Day 4 Lactation
0	10	3	7	0	21.7	10	100	13.4 ± 3.6	12.5 ± 3.5	12.4 ± 3.5	12.3 ± 3.3	12.3 ± 3.3
1000	10	4	6	0	21.6	10	100	15.4 ± 2.5	14.7 ± 2.5	14.7 ± 2.5	14.0 ± 3.9	13.1 ± 4.7
5000	10	4	5	1	21.7	10	100	14.2 ± 3.8	13.1 ± 3.4	13.1 ± 3.4	13.0 ± 3.4	13.0 ± 3.4
20 000	8	2	6	0	21.8	8	100	12.1 ± 1.5	11.8 ± 1.4	11.4 ± 1.7	11.4 ± 1.7	11.4 ± 1.7

SD = Standard Deviation

 

Table 7 Group Mean F1 Survival Indices

Dose Level (ppm)	Birth Index			Live Birth Index			Viability Index Days 1 to 4
	Mean Litter Index (%)	Number Losing >2 Pups	Number of Litters	Mean Litter Index (%)	Number Losing >1 Pups	Number of Litters	Mean Litter Index (%)	Number Losing >3 Pups	Number of Litters
0	90	0	9	99	0	10	99	0	10
1000	96	0	10	100	0	10	88	1	10
5000	93	0	10	100	0	10	99	0	10
20 000	97	0	8	97	1	8	100	0	8

 

Table 8 Group Mean Litter and Pup Weight During Lactation (g) ± Standard Deviation

Dose Level (ppm)	Litter		Mean of Litter Mean Pup Weight
	Day 1	Day 4	Males		Females
			Day 1	Day 4	Day 1	Day 4
0	84 ± 17	126 ± 23	7.2 ± 0.9	10.8 ± 2.0	6.9 ± 1.2	10.5 ± 2.2
1000	85 ± 21	124 ± 50	6.4 ± 0.9	9.7 ± 1.9	5.9 ± 0.8	9.0 ± 2.6
5000	82 ± 16	121 ± 22	6.7 ± 1.2	10.1 ± 2.2	6.2 ± 1.0	9.4 ± 1.7
20 000	79 ± 9	117 ± 12	7.2 ± 0.8	10.7 ± 1.4	6.8 ± 0.6	10.2 ± 1.2

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, toxicity was exhibited at levels of 5000 and 20 000 ppm, but there were no clear effects of toxicity at 1000 ppm. Therefore the parental repeated dose NOEL was considered to be 1000 ppm. For reproductive parameters the NOEL was considered to be 5000 ppm.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guideline OECD 422 under GLP conditions.

Four groups of 10 male and 10 female Sprague-Dawley rats received the test material via the diet at concentrations of 0, 1000, 5000 and 20 000 ppm. The males were dosed for at least 4 weeks, starting 2 weeks prior to mating. The females were dosed from 2 weeks prior to mating until at least day 6 of lactation.

The animals were monitored for clinical signs, bodyweight, food consumption, mating and litter performance. Haematology and clinical chemistry parameters were investigated; additionally, all animals were subjected to necropsy and histopathological investigations were carried out on animals in the control and 20 000 ppm dose groups. The pups were weighed and examined for gross external abnormalities.

 

At 20 000 ppm, in-life observations included decreased weight gain and food consumption in both sexes. Increased male liver weight following covariance analysis, and increases in bilirubin and alkaline phosphatase were noted in both sexes. In addition, small decreases were noted in adrenal gland weight in both sexes, and in albumin, white blood cell count and ovary weight in females; spleen weight and cholesterol were slightly increased in males.

At 5000 ppm, alkaline phosphatase levels in both sexes were increased. Female adrenal gland weight was reduced.

The only indication of reproductive toxicity was a marginal decrease in implant sites at 20 000 ppm with a corresponding decrease in the mean total number of pups born compared to all other dose groups. However, due to the differences from the control being slight, there is some doubt as to the reproducibility of this finding.

Under the conditions of this study, toxicity was exhibited at levels of 5000 and 20 000 ppm, but there were no clear effects of toxicity at 1000 ppm. Therefore the parental repeated dose NOEL was considered to be 1000 ppm. For reproductive parameters the NOEL was considered to be 5000 ppm.