Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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
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
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.
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
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
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.
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.
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)
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
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.
Reproductive effects observed:
not specified

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

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Two available studies were conducted in accordance with the standardised guideline OECD 422 under GLP conditions and were assigned a reliability score of 2 in accordance with the criteria set forth by Klimisch (1997). A pre-natal study was also available for DEA, which was assigned a Klimisch = 2.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No reproductive or developmental studies are available on the target substance. However, according the TIMES rat liver S9 metabolism simulator, the target substance is fully metabolized into tall oil and diethanolamine (DEA). Therefore, data on these two source substances are used to fulfill the reproductive endpoints. Further discussion on the metabolism of the target substance is given in the Read Across Justification Document in Section 13.

Effect on fertility: via oral route

The first combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the read across material tall oil 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, small decreases were noted in ovary weight in females; 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, for reproductive parameters the NOEL was considered to be 5000 ppm, equivalent to 500 mg/kg bw/day.

 

The second combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the read across material tall oil, compound with ethanolamine in accordance with the standardised guideline OECD 422 under GLP conditions.

Three groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats were dosed with the vehicle (UHP water) following the same dosing regimen as the treated animals and were used as controls. The males were dosed for 14 days prior to pairing, during pairing and until the day prior to necropsy. The females were dosed for 14 days prior to pairing, during pairing and gestation, and up to and including Day 3 of lactation.

There were no unscheduled deaths on the study and administration of the test material was well tolerated; there was no effect of treatment on fertility or mating performance. There was no effect of treatment with the test material on the mean duration of gestation. The mean number of pups born per litter, sex ratio and mean live birth, viability and cumulative survival indices in all treated groups were comparable with controls. There were no unscheduled pup deaths on the study.

Group mean pup body weight gains between Days 1 and 4 of age were unaffected by maternal treatment with the test material and there were no necropsy findings for pups considered to be related to maternal treatment with the test material.

Under the conditions of this study, in the Wistar strain rat, the No Observed Adverse Effect Level (NOAEL) for male and female reproductive performance was considered to be 1000 mg/kg/day.

 

In accordance with Section 1 of Annex XI of the Regulation (EC) 1907/2006 (REACH), it is considered justified to omit the two-generation reproductive toxicity study (required in section 8.7.3 of Annex IX) as testing does not appear scientifically necessary.

Two oral studies conducted to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) for tall oil and tall oil, compound with ethanolamine already sufficiently address the reproductive toxicity data requirements. No effects on reproductive function were seen in the screening studies and the data available is sufficient to allow for the derivation of DNEL values.

 

Furthermore, in accordance with the general principles of Annex XI of Regulation (EC) 1907/2006 (REACH), it is considered scientifically justified to omit further testing for toxicity to reproduction by the most appropriate route of exposure on the diethanolamine component of the registered substance. It is considered that further animal testing on this substance is unlikely to add substantial value to the dataset and as such is not proposed by the registrant at this time.


Short description of key information:
TOXICITY TO REPRODUCTION
NOEL tall oil 500 mg/kg/day male and female Sprague-Dawley strain rats
NOAEL tall oil, compound with ethanolamine 1000 mg/kg/day male and female Wistar strain rats

Justification for selection of Effect on fertility via oral route:
This endpoint was addressed based on the metabolism of the target substance as seen with the TIMES rat liver S9 simulator. The two metabolites are tall oil and diethanolamine (DEA). More information is given in the Read Across Justification document in Section 13. The endpoint is addressed with two combined repeated dose toxicity study with reproduction/developmental toxicity screening studies being carried out on the read across materials tall oil and tall oil, compound with ethanolamine. An OECD 414 study is also available for DEA.
The study conducted on tall oil was selected as key as a precaution on the basis that the NOEL achieved in this study was lower than the NOAEL reported for the study on tall oil, compound with ethanolamine.
It is considered that these studies together are an accurate reflection of the total composition of the registered substance, Tall Oil, compound with diethanolamine.
Therefore, the extended one generation reproductive toxicity study is not scientifically justified and is being waived.

Effects on developmental toxicity

Description of key information
DEVELOPMENTAL TOXICITY / TERATOGENICITY 
NOAEL tall oil 2000 mg/kg/day Sprague-Dawley strain rats in 422 screening study (oral)
NOAEL tall oil, compound with ethanolamine 1000 mg/kg/day Wistar strain rats in 422 screening study (oral)
Teratogenicity NOAEC for diethanolamine was determined to be ≥0.2 mg/L air in Wistar strain rats in a 414 study (inhalation)
NOEL maternal rat = 150 mg/kg bw/day in a pre-natal dermal study
NOEL developmental rat = 380 mg/kg bw/day in a pre-natal dermal study
NOEL maternal rabbit = 35 mg/kg bw/day in a pre-natal dermal study
NOEL developmental rabbit = 350 mg/kg bw/day in a pre-natal dermal study
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2 August 2012 to 12 November 2012
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.
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI(Han)
- Age at study initiation: On the first day of dosing, animals were 71 to 78 days of age.
- Weight at study initiation: On the first day of dosing, the males weighed 283 g to 333 g and the females weighed 184 g to 219 g.
- Housing: The animals were housed in groups of 5 by sex until pairing and for males post-pairing. For pairing, one male and one female were housed together and mated females were housed individually during gestation and with their litter during the lactation period. Males were housed in grid-floor cages suspended over paper-lined trays. During the pre-pairing and mating periods females were housed in grid-floor cages suspended over paper-lined trays and following mating females and their litter were housed in solid-floor cages with appropriate bedding provided.
- Diet (e.g. ad libitum): A pelleted rodent diet was supplied ad libitum.
- Water (e.g. ad libitum): Mains tap water (in bottles) was freely available.
- Acclimation period: The animals were acclimatised within the study room for 14 days before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C (target range 21 ± 2 °C).
- Humidity (%): 46 to 79 % (target range 55 ± 15 %).
- Air changes (per hr): Not reported - the room was air-conditioned.
- Photoperiod (hrs dark / hrs light): The study room was illuminated by fluorescent light set to give a cycle of 12 hours light and 12 hours dark.

IN-LIFE DATES: From: 2 August 2012 To: 12 November 2012
Route of administration:
oral: gavage
Vehicle:
other: Elga UHP water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The required amount of test material was weighed and approximately 80 % of the vehicle was added. The mixture was stirred and heated to approximately 50 ± 5 °C, as required, until a visibly homogenous liquid was formed. The formulation was allowed to cool to ambient temperature, followed by further addition with the vehicle to the exact volume. Due to the lack of any stability information, the formulations were prepared daily and used within 3 hours of preparation.

VEHICLE
- Dose volume: 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
FORMULATION SAMPLING
Samples were taken from each formulation. The formulations (including controls) prepared for use on the first day of dosing and subsequent formulations prepared for use during Week 6 were analysed to assess achieved concentrations. All remaining samples were stored frozen (approximately -18 °C).

ANALYSES OF TEST MATERIAL FORMULATIONS
Samples from each designated formulation, including the vehicle used to dose the control group, were analysed for the test material. During Week 6, individual achieved concentrations from the formulations prepared for use on Day 40 of the study (Groups 2 to 4) ranged from 113 to 126 % of the nominal. Since the majority of individual concentrations were above the acceptance criterion and contingency samples could not be analysed due to a lack of the frozen stability data, analysis of test material formulations prepared for use on Day 41 of the study (Groups 2 to 4 only) was performed.

METHOD OF ANALYSIS
The analytical procedure for the determination of the test material in water was conducted using UV/visible spectrophotometry using the test material, water and HPLC grade methanol.
- Instrument: Unicam UV/visible spectrophotometer

PROCEDURE
Linearity of Detection
The linearity of detection was assessed from triplicate measurements of six standards prepared at concentrations over the calibration range of the assay. The standard concentration and response data were subjected to linear regression analysis and the assay considered linear if the correlation coefficient (r) was no less than 0.99.
The linear correlation coefficient over the calibration range 20.32 to 81.80 µg/mL of the test material was 0.9968.

Precision of Detection
The precision of detection was assessed over the linear range from the coefficient of variation of the response factors (response/concentration) of all standards analysed in triplicate.
The precision of detection at the mid-point of the linear range was assessed from the coefficient of variation of the responses of a mid-point standard analysed six times consecutively. The precision of detection was considered acceptable if the coefficients of variation were no greater than 7.5 % for each assessment.
The precision of detection over the linear range was 3.4 %. The precision of detection at the mid-point of the linear range was 0 %.

System Stability
The stability of the system was assessed from the coefficient of variation of the responses of a mid-point standard, analysed six times interspersed throughout the run, and was considered acceptable if no greater than 7.5 %.
The stability of the system was 0.1 %.

Specificity
The specificity of the assay was assessed from the analysis of control samples and reagent blanks and was considered acceptable if the absorbance was no greater than 5 % of that of the lowest concentration standard.
No response that was attributed to the test material was observed in the control samples or reagent blanks.

Intra-Assay Accuracy and Precision
The intra-assay accuracy at each concentration, expressed as the mean accuracy values (n=3) was assessed from the analysis of triplicate samples of the test material in water, at concentrations of 10 mg/mL and 200 mg/mL. The assay was considered accurate if the individual and the mean measured concentrations of the test material were between 85 and 115 % of their nominal values. The intra-assay precision, expressed as the coefficient of variation of the individual accuracy values at each concentration, was considered acceptable if no greater than 10 %.
The individual accuracy values at 10 mg/mL were between 85 and 89 % with a mean value of 87 % and a precision value of 2.5 %. The individual accuracy values at 200 mg/mL were between 85 and 87 % with a mean value of 86 % and a precision value of 1.1 %.

Inter-Assay Accuracy and Precision
Assay accuracy and precision, were repeated on a separate occasion, using freshly prepared solutions and reagents, to establish the effects of such changes.
The inter-assay accuracy at each concentration, expressed as the mean accuracy values (n=6) obtained over two occasions, was considered acceptable if the individual and the mean measured concentrations of the test material were between 85 and 115 % of their nominal values.
The inter-assay precision at each concentration, expressed as the coefficient of variation of the individual accuracy values (n=6) obtained over two occasions, was considered acceptable if no greater than 15 %.
The individual accuracy values at 10 mg/mL were between 85 and 95 % with a mean value of 90 % and a precision value of 4.3 %. The individual accuracy values at 200 mg/mL were between 85 and 89 % with a mean value of 88 % and a precision value of 2.0 %.

RESULTS OF FORMULATION ANALYSIS
Formulations used to dose animals during Week 1 of the study were considered accurate since the measured concentrations were within 15 % of their nominal values which fulfilled the acceptance criterion.
The majority of samples from test formulations used to dose animals in Groups 2, 3 and 4 on Day 40 of the study (Week 6) showed higher levels of the test material than the target values (by up to 26 %), which was outside the specified acceptance criterion. Test material formulations used to dose animals in Groups 2, 3 and 4 on Day 41 of dosing were also higher than their nominal values (by up to 30 %), which was again outside the specified acceptance criterion. Although no reason could be established for these higher values, considering these animals were dosed at higher dose levels than their target, this was considered to have no impact on the validity of the No Observed Adverse Effect Levels (NOAELs) determined in this study.
No test material was detected in vehicle used to dose animals in Group 1.
Details on mating procedure:
- M/F ratio per cage: After the pre-pairing dosing period, each female was paired with a treated male from the same group.
- Length of cohabitation: 14 days.
- Proof of pregnancy: During the pairing period, vaginal smears were taken daily, by lavage, until mating was confirmed either by sperm being found in the smear or by the number and nature of copulation plugs. The smear was examined under light microscopy and the stage of the oestrous cycle was determined by the type of cell present. The number of copulation plugs was also recorded to give an assessment of the mating activity of the animals. The day on which sperm were detected was referred to as Day 0 of gestation with the next day classified as Day 1 of gestation.
- After successful mating each pregnant female was caged (how): Mated females were housed individually during gestation and with their litter during the lactation period.
Duration of treatment / exposure:
The males were dosed for 14 days prior to, and during pairing, and until the day prior to necropsy. The females were dosed once daily for 14 days prior to, and during pairing, gestation and until Day 3 of lactation, inclusive.
Any female in mid-parturition at the time of dosing was not dosed on that day.
Frequency of treatment:
Once daily
Duration of test:
Females were sacrificed on Days 41 to 45; males were sacrificed on Day 55.
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 results from a preliminary study. A high dose level of 1000 mg/kg/day was chosen, as this is the maximum dose level required by the guidelines. At this dose level in the preliminary study, clinical signs remained confined to excessive pre and/or post-dose salivation.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined twice daily for mortality and morbidity. From the start of treatment, all animals were examined daily for clinical signs of toxicity or changes in behaviour and appearance.
During the treatment period, each main study animal was routinely checked pre-dose and soon after completion of dosing. On week days, additional observations were made approximately 1 hour after dosing and either approximately 4 hours after dosing or at the end of the working day (whichever was sooner). At weekends and public holidays, additional observations were made approximately 1 hour after dosing or at the end of the working day (whichever was sooner).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed clinical examination once each week, from the start of treatment. Standard arena observations (functional observations) were made cage side and outside the home cage, in a standard arena, for all animals. Observations were recorded once prior to the start of dosing and once weekly thereafter during the dosing period, at approximately the same time of day on each occasion (afternoon). On Day 40 of the study, these observations were not recorded for any females in mid-parturition or on Day 0 of lactation; they were instead recorded on Day 42 of the study for those females.

BODY WEIGHT: Yes
- Time schedule for examinations: Female body weights were recorded at the start of treatment and then at weekly intervals until the day of mating. Body weights for females were also recorded on Days 0, 7, 14 and 20 of gestation and then on Days 0 (where required for dose volume calculation), 1 and 4 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule: The amount of food consumed by the animals in each cage was recorded at weekly intervals during the pre-pairing dosing period. Food consumption of the females was also recorded over Days 0 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 20 of gestation and over Days 1 to 4 of lactation.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

URINALYSIS: No

OTHER: Neurobehavioural examinations, haematology and coagulation parameters and clinical chemistry parameters were evaluated.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes. The number of implantation scars and the number of corpora lutea for each female was recorded.
Fetal examinations:
F1 GENERATION
- Litter size and sex: The total litter size was recorded after completion of parturition and daily thereafter. Numbers of each sex were recorded on the same day as body weights were collected. The percentage of male pups, out of the total number of pups, was calculated for each litter. Day 0 of lactation was the day of completion of littering. The following day was considered to be Day 1 of lactation.
- Clinical observations: All pups were examined daily for clinical signs of toxicity or changes in behaviour and appearance.
- Mortality: Animals were examined twice daily for mortality and morbidity.
- Bodyweights: Pups were weighed individually on Days 1 and 4 of age.
Statistics:
Non-parametric methods were employed if it was considered that the assumptions required for parametric analysis did not hold.
No analyses were performed for variables for which there were less than five independent observations in the control group; when treated groups had fewer than five observations analyses were performed at the discretion of the statistician.
Data from each sex was analysed separately. Statistical significance was declared at the p<0.05 level in all cases and noted at the p<0.01 and p<0.001 levels.

COMPARISONS
Group 1 (control) against Groups 2, 3 and 4 (100, 300 and 1000 mg/kg, respectively)

STATISTICAL TESTS AND PARAMETERS
Data were processed to give group mean values and standard deviations where appropriate. Where the data allowed, the following methods were used for statistical analysis:

ANALYSIS OF VARIANCE AND WILLIAMS' TEST
(Non-parametric alternative: Kruskal-Wallis ANOVA and Shirley's Test)
Bodyweights and bodyweight gains, food consumption, number of implantation scars and number of corpora lutea, number of pups born, pup body weights and body weight gains (by sex and on litter basis), percentage of male pups, time course of mating, number of copulation plugs, duration of gestation, organ weights (absolute), relative organ weights (organ weights as a percentage of terminal body weight), clinical pathology data, grip strength and motor activity (timepoints 1 and 1 - 6 (total)).

ANALYSIS OF COVARIANCE
Motor activity (timepoints 2 to 6)

ANALYSIS OF VARIANCE
(Non-parametric alternative: Kruskal-Wallis Test)
Pre-dose body weights (Day 1)

COCHRAN ARMITAGE TREND TEST
Copulation and fertility indices

JONCKHEERE TERPSTRA TEST
Litter survival indices
Indices:
REPRODUCTIVE INDICES
Copulation index (%) = (number of animals mated / number of animals paired) x 100
Female fertility index (%) = (number of pregnant animals / number of animals mated) x 100
Male fertility index (%) = (number of males siring a pregnancy / number of paired males )x 100
Gestation index (%) = (number of pregnant females with live pups born / number of pregnant females) x 100

OFFSPRING VIABILITY INDICES
Live birth index (%) = (number of pups born alive / total number of pups born) x 100
Viability index 1 (%) = (number of pups alive Day 4 of age / number of pups born alive) x 100
Cumulative survival index (%) = (number of pups alive Day 4 of age / total number of pups born) x 100
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There was no mortality and there were no clinical signs considered to be related to the toxicity of the test material.

BODY WEIGHT AND FOOD CONSUMPTION
During the first week of the pre-pairing period, females given 1000 mg/kg/day showed a stasis in group mean bodyweight with half of these animals losing body weight; in comparison control animals gained weight during this period and the difference was statistically significant (p<0.001). Thereafter, an improvement was evident such that during the second week of dosing, the mean bodyweight gain for these females was higher than controls, albeit without achieving statistical significance. Group mean bodyweight gains for these females during the gestation and lactation periods remained unaffected. For females receiving 100 or 300 mg/kg/day of the test material, there was no effect of treatment on body weight gain during any stage of the study.

Females given 1000 mg/kg/day showed slightly lower food intake during the pre-pairing period and over Days 0 to 4 of gestation (p<0.05) when compared with controls. Throughout the remainder of the gestation period, the mean food intake values for this group of females were marginally lower than controls, albeit without achieving statistical significance. During lactation, food intake values for these females were similar to controls.
For animals given 100 or 300 mg/kg/day of the test material, food intake values were generally similar to controls throughout the study.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE
There was no effect of treatment with the test material on the mean number of days taken to mate. With an exception of one female from the control group and one female from the group receiving 300 mg/kg/day, all other females mated within 4 days of pairing; the two exceptions were in persistent dioestrous prior to mating. The mean number of copulation plugs remained unaffected by treatment with the test material.

REPRODUCTIVE PERFORMANCE
There was no effect of treatment on fertility or mating performance.
There was no effect of treatment on the mean duration of gestation.
At 1000 mg/kg/day, group mean value for implantation scars was marginally but statistically significantly lower (p<0.05) than controls but this was considered coincidental; only 2 out of 10 animals had individual values slightly below controls. The mean number of corpora lutea for this group of females was also statistically significantly lower (p<0.05) than controls, however the recording of corpora lutea on Day 4 of lactation is considered inaccurate. Since post-partum oestrous occurs 24 to 48 hours after parturition in rats, during the lactation period there are two sets of corpora lutea; one from pregnancy and the other from the new oestrous. Since corpora lutea are counted under low magnification, it is impossible to distinguish between the two, and counting the corpora lutea may result in inaccurate numbers.

ORGAN WEIGHTS
At 1000 mg/kg/day, absolute and bodyweight-related liver weights were statistically significantly higher than controls (p<0.01). The majority of corresponding individual bodyweight-related liver weights for females, including controls, were above the background control data ranges. In the absence of any histopathology findings for the liver, the increases in liver weight were considered to be adaptive in nature and not indicative of toxicity.
At 300 or 1000 mg/kg/day, females also showed statistically significantly lower body weight-related heart weights than controls (p<0.05) but without any dose-relationship. The majority of individual values were within the background control data ranges and as there were no corresponding histopathology findings, this finding was considered not to be related to treatment with the test material.

GROSS PATHOLOGY
There were no macroscopic findings at necropsy considered to be related to the treatment with the test material.

HISTOPATHOLOGY
There was an equivocal change (erythrocytosis/erythrophagia) in the mesenteric lymph nodes for animals receiving 1000 mg/kg/day. No effect was noted for these parameters in the low or intermediate dose groups. In addition, an assessment of the bone marrow smears from control animals and those given 1000 mg/kg/day indicated that there was no effect of treatment on the assessed parameters.
A variety of spontaneous changes was noted in control and treated animals with no indication of an effect of treatment. The spectrum of these findings is generally consistent with changes encountered in rats of this age kept under laboratory conditions.

OTHER FINDINGS
HAEMATOLOGY AND COAGULATION
Leucocyte and lymphocyte values in all female groups given the test material were slightly lower than the respective control group, albeit without achieving statistical significance. In the absence of any associated histopathology findings, this was considered not to be of toxicological significance.
At 1000 mg/kg/day, females showed a marginal but statistically significant lowering in haemoglobin concentration (p<0.05) and slight prolongation of prothrombin time (p<0.001) when compared with controls. Individual haemoglobin concentrations were within the background control data range but respective prothrombin times were above these ranges. In the absence of any associated histopathology changes these findings were considered not to be toxicologically significant.

BLOOD CHEMISTRY
Statistically significantly higher mean plasma concentration of bile acids (p<0.05) was observed in females given 1000 mg/kg/day with respect to controls. At 100 or 300 mg/kg/day, a small number also showed higher concentrations of bile acids than controls. High individual variation seen in these animals may be due to the fact that these animals were not fasted prior to bleeding and the intergroup differences were considered unrelated to treatment.

FUNCTIONAL OBSERVATIONS
There were no clear effects of treatment on functional observations monitored throughout the study or sensory reactivity to visual, acoustic, tactile or proprioceptive stimuli monitored towards the end of the study.
- Grip Strength: Any differences between control and treated animals were non dose-related and individual variation was high amongst animals. These differences were considered not related to treatment with the test material.
- Motor Activity: Any differences between control and treated animals were non dose-related and individual variation was high amongst animals. These differences were considered not related to treatment with the test material.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The mean number of pups born per litter, sex ratio and mean live birth, viability and cumulative survival indices in all treated groups were comparable with controls.
There were no unscheduled pup deaths on the study. Group mean pup body weight gains between Days 1 and 4 of age were unaffected by maternal treatment with the test material.
A variety of clinical signs noted across all groups, including controls, were considered unrelated to maternal treatment with the test item.
There were no necropsy findings for pups considered to be related to maternal treatment with the test material.
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1 F1 Pup Bodyweights (g) - Group Mean Values

Males

Dose (mg/kg/day)

N

Bodyweight(g)

Bodyweight Gain(g)

(Day 1 to 4)

1 Day of Age

4 Days of Age

0

10

6.9 ± 0.5

10.5 ± 0.7

3.7 ± 0.3

100

10

7.2 ± 0.9

11.0 ± 1.3

3.7 ± 0.5

300

10

6.5 ± 0.6

9.8 ± 0.9

3.3 ± 0.4

1000

10

7.0 ± 1.0

10.6 ± 1.6

3.5 ± 0.7

Females

Dose (mg/kg/day)

N

Bodyweight(g)

Bodyweight Gain(g)

(Day 1 to 4)

1 Day of Age

4 Days of Age

0

10

6.5 ± 0.3

10.1 ± 0.6

3.6 ± 0.4

100

10

6.9 ± 0.8

10.6 ± 1.2

3.7 ± 0.5

300

10

6.2 ± 0.7

9.5 ± 1.1

3.3 ± 0.5

1000

10

6.7 ± 0.9

10.2 ± 1.3

3.6 ± 0.5

= Statistically analysed

N = Number of litters in mean

 

Table 4 pup Necropsy - F1 Generation

Parameter

Dose (mg/kg/day)

0

100

300

1000

N

10

10

10

10

Number of Pups

Born

Found dead/killed prematurely

Missing (presumed cannibalised)

Schedule sacrificed

123

0

0

123

118

0

0

118

129

0

0

129

112

0

0

112

Findings: Dead/Moribund/Sacrificed Pups

No abnormalities

No milk in stomach

Right testis & epididymis abnormal red colour

123

0

0

117

1

0

129

0

0

109

2

1

N = Total number of litters

Conclusions:
Under the conditions of this study, in the Wistar strain rat, the No Observed Adverse Effect Level (NOAEL) for male and female repeated-dose toxicology was considered to be 300 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) for developmental effects was considered to be 1000 mg/kg/day.
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.

Three groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats were dosed with the vehicle (UHP water) following the same dosing regimen as the treated animals and were used as controls. The males were dosed for 14 days prior to pairing, during pairing and until the day prior to necropsy. The females were dosed for 14 days prior to pairing, during pairing and gestation, and up to and including Day 3 of lactation.

There were no unscheduled deaths on the study and administration of the test material was well tolerated; some parental effects were seen on bodyweights, lymphocyte counts and bone marrow adipocytes however the magnitude of change was small.

There was no effect of treatment on fertility or mating performance. There was no effect of treatment with the test material on the mean duration of gestation. The mean number of pups born per litter, sex ratio and mean live birth, viability and cumulative survival indices in all treated groups were comparable with controls. There were no unscheduled pup deaths on the study.

Group mean pup body weight gains between Days 1 and 4 of age were unaffected by maternal treatment with the test material and there were no necropsy findings for pups considered to be related to maternal treatment with the test material.

Under the conditions of this study, in the Wistar strain rat, the No Observed Adverse Effect Level (NOAEL) for male and female repeated-dose toxicology was considered to be 300 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) for developmental effects was considered to be 1000 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
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.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
- Source: Dr. K Thomae GmbH, Biberach Germany
- Age at study initiation: 68-70 days
- Weight at study initiation: ca. 214 g
- Housing: individual in wire mesh cages
- Diet: Kliba rat/mouse laboratory diet, Klingenthalmuehle AG Kaiseraugst, Switzerland ad libitum
- Water: Tap water ad libitum
- Acclimation period: 5 days
- Adaption period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
nose/head only
Vehicle:
unchanged (no vehicle)
Details on exposure:
Test group 1
The substance to be tested was supplied to a two component atomizer by means of a continuous infusion pump and was atomized with compressed air. Having passed a glass separator, the liquid aerosol was diluted with blast air (about 50% rel. humidity; 22°C) and part of the air stream was supplied to the exposure apparatus (INA 60). The remainder of the aerosol generated was supplied to the exhaust air equipment (exhaust air 2).

Test group 2
The substance to be tested was supplied to a two component atomizer by means of a continuous infusion pump and was atomized with compressed air. Having passed a glass separator, the liquid aerosol was diluted with blast air (about 50% rel. humidity; 22°C) and the air stream was supplied to the exposure apparatus (INA 60).

Test group 3
The substance to be tested was supplied to a two component atomizer out of a storage vessel by means of a diaphragm metering pump and was atomized with compressed air . Having passed a glass separator, the liquid aerosol was diluted with blast air (about 50% rel. humidity; 22°C) and the air stream was supplied to the exposure apparatus (INA 60).

As the test substance is sensitive to oxygen, the open storage vessel of the diaphragm pump was kept covered with a nitrogen cushion (5 l/h).

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of the atmospheres in test groups 1 and 2 was monitored by means of light scattering photometers. The light scattering photometers were calibrated with mixtures of the test substance and air that were generated in the exposure apparatus not containing animals according to the test group concentrations.
The concentration in test group 3 was analyzed by a corrected gravimetrical determination which was calibrated with an indirect photometric method.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1-4
- Length of cohabitation: 4 pm - 7.30 am
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy:sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
gestation day 6 - 15
Frequency of treatment:
6h/day
Duration of test:
gestation day 6-20 (gd 16-20: post exposure observation period)
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during the preflow and the observation period, 3 times per day during the exposure period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily during the preflow and the observation period, 3 times per day during the exposure period

BODY WEIGHT: Yes
- Time schedule for examinations: 0, 3, 6, 9, 12, 15, 18, 20 p.c.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 (gross pathology)


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

- Weight of uterus before it was opened
- Number of corpora lutea
- Number and distribution of implantation sites classified as :
• live fetuses
• dead implantations:
a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy)
b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

Furthermore calculations of conception rate and pre- and postimplantation losses were carried out :
- The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals/ number of corpora lutea) x100

-The postimplantation loss (in %) was calculated* from the following formula:
(number of implantations - number of live fetuses)/ number of implantations x100

* Calculation on the basis of each individual pregnant animal with scheduled sacrifice
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
- Dunnett's Test: body weight, body weight change, corrected body weight  gain (net maternal body weight change), weight of the uterus before it  was opened, weight of fetuses, weight of placentae, corpora lutea,  implantations, pre and postimplantation losses, resorptions and live  fetuses.
- Fisher's Exact Test: conception rate, mortality (of the dams) and all  fetal findings.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No deaths or treatment related finding based on body weight, corrected body weight gain, necropsy findings, uterus weight, reproduction data of the dams.
Clinical findings:
During the preflow period the animals of all showed no clinical signs and findings but during the exposure period eight animals of the highest concentration group (0.2 mg/L) showed bloody discharge from the vagina on day 14 p.c. after exposure.
Dose descriptor:
NOAEC
Effect level:
0.05 mg/L air
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
EXAMINATION OF FETUSES:
- Sex distribution: Not affected in any group.
- Placenta weights: Not affected in any group.
- Weight of fetuses Not affected in any group.
- External, soft tissue and skeletal fetal observations:
The only substance-related finding was the increased occurrence of high dose fetuses with rudimentary cervical rib(s). Due to the high frequency of this finding in the 0.2 mg/L fetuses, the incidence of skeletal variations was statistically significantly increased.

All other differences between the control group and the substance-treated groups concerning fetal external, soft tissue and/or skeletal observations were without biological relevance and/or appeared to about the same extent as in the historical control data.
Dose descriptor:
NOAEC
Effect level:
>= 0.2 mg/L air
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Results summary:

The exposure of pregnant female Wistar to an aerosol of Diethanolamine in a head/nose exposure systems for 6 h/day on day 6 through day 15

post coitum at concentrations of 0; 0.01; 0.05; 0.2 mg/L (0; 10, 50, 200 mg/m³) led to signs of maternal toxicity at the highest concentration

(0.2 mg/L).

Maternal toxicity was substantiated by adverse clinical symptoms (vaginal hemorrhages) in 8 of the 21 pregnant rats on day 14 p.c. At this dose level a markedly increased number of fetuses with skeletal variations (mainly cervical rib(s)) were also recorded but substance-related teratogenic

effects were not detected in any fetus. Thus, signs of prenatal developmental toxicity did only occur at a maternal toxic concentration. There were no adverse effects on dams or fetuses at the low or mid concentrations (0.01 or 0.05 mg/L).

The NOAEC for maternal and prenatal developmental toxicity was 0.05 mg/L (50 mg/m³), the NOAEC for teratogenicity was >0.2 mg/L (200 mg/m³).

Detailed results:

Concentrations:                    

Target

Measured

MMAD      

% aerosol

0.01 mg/L

10.0 mg/m³        

1.2 µm        

100 %

0.05 mg/L

50.2 mg/m³ 

0.4 µm        

98 %

0.2 mg/L

202 mg/m³        

0.6 µm        

98 %



 

Maternal, litter, fetal data - prenatal developmental toxicity  (inhalation) study in Wistar rats

mg/L

0

0.01

0.05

0.2

n (females)

25

25

25

25

Pregnant

21

21

23

23

Mortality

0

0

0

0

Body weight gain GD6-15 (g)

71

71

77

70

NWC from GD 6

38

43

39

41

Gravid uterus (g)

71

68

76

67

Corpora lutea (mean)                

14.5

15.3

15.2

14.6

Implantation  (mean)                

13.2

13.2

13.9

12.5

Post implantation loss

5.5

10.0

4.9

10.2

Resorption  (mean) 

0.8

1.4

0.7

1.3

Live fetuses (mean)                

12.5

11.8

13.2

11.2

Placental weight (g)

0.44

0.44

0.43

0.43

Fetus evaluated

262

248

303

258

Litter evaluated

21

21

23

23

Fetal weight (g) (males/females)        

4.0/3.8        

4.0/3.8

4.0/3.8        

4.1/3.9

Total external malformations (fetus/litter)

0/0        

1/1        

0/0        

0/0        

Total external variations (fetus/litter)

0/0        

0/0        

0/0        

0/0        

Total soft tissue malformations (fetus/litter)        

1/1        

2/2        

2/2        

0/0

Total soft tissue variations (fetus/litter)        

20/9

20/15        

25/16        

16/10

Total skeletal malformations (fetus/litter)        

5/5

5/4        

7/6        

5/5

Total skeletal variations (fetus/litter)        

59/19        

58/20        

69/22        

78/22*

GD = gestation day, NWC = net body weight change, 
* p<0.05 

**p<0.01

Conclusions:
Under the conditions of this study, the NOAEC for teratogenicity was determined to be ≥0.2 mg/L air.
Executive summary:

The potential of the test material to cause developmental effects was investigated in accordance with the standardised guideline OECD 414 under GLP conditions.

Female Wistar rats were exposed to the test material via the inhalation route at concentrations of 0.01, 0.05 and 0.2 mg/L (25 animals per dose level) from day 6 of gestation to day 15.

Under the conditions of this study, the NOAEC for teratogenicity was determined to be ≥0.2 mg/L air, whilst the NOAEC for maternal toxicity was determined to be 0.05 mg/L air.

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
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.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
- Source: Charles River Laboratory (Portage, MI)
- Age at study initiation: 68-70 days
- Weight at study initiation: ca. 209 - 251 g
- Housing: individual in wire mesh cages
- Diet: Ground certified Rodent Diet (RMH 3200, agway, Inc, Waverly, NY) available ad libitum
- Water: Tap water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 66-77
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
dermal
Vehicle:
water
Details on exposure:
The test substance was dermally applied daily directly to the backs (clipped free of hair) of rats at a constant volume of 4 ml/kg/day. DEA remained in contact with the skin for 6 hours per day at doses of 0, 150, 500 or 1500 mg/kg bw/day from gestation day (GD) 6 through to 15. Following administration the dosing site was covered by sterilized gauze and then further occluded with polyvinyl fil attached to a specifically designed Lycra-Spandex jacket with velcro closures. Approximatley 6 hours after dosing, the jacket and gauze were removed, and the dosing site was wiped gently with gauze dampened with warm water and blotted dry.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration and homogenicity of the DEA dosing solution was verified using a Hewlett-Packard 5880A gas chromatograph with a flame ionization detector. DEA concentrations ranged fro, 100.7 to 101.7% of the target. Rats in various dose groups did not receive the total volume of DEA during dosing on GD 12-15. There was a possible 10-25 % deficit in the expected doses delivered with the greatest difference seen in the 500 mg/kg bw/day dose group on GD 13.
Details on mating procedure:
Virgin female rats weighing between 209-251 g were mated overnight with male rats (one female:one male). Evidence of vaginal or dropped copulation plugs was considered a sign of successful mating and the day on which such evidence was found was designated as GD 0. Mated females were housed singly in stainless-steel wire-mesh cages.
Duration of treatment / exposure:
6 hours / day
Frequency of treatment:
Daily
Duration of test:
From GD 6-15 inclusive
No. of animals per sex per dose:
25 successfully mated females were assigned to each dose group
Control animals:
yes
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for evidence of skin irritation and clinical signs during the dosing period and once daily during the post-treatment period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: wice daily for evidence of skin irritation and clinical signs during the dosing period and once daily during the post-treatment period

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 3, 6, 9, 12, 15, 18, 21 p.c.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 (gross pathology)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:

- Weight of uterus before it was opened
- Number of corpora lutea
- Number and distribution of implantation sites classified as :
• live fetuses
• dead implantations:
a) early resorptions - Uteri from females that appeared to be nongravi were placed in a 10% ammonium sulfide solution for detection of early resorptions (Salewski, 1964)



Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No data
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Continuous variables were compared for homogenicity of variance using Levene's test for equal variance. Based on the outcome of this analysis, a parametric or non-parametric analysis of variance (ANOVA) was performed as appropriate. Significant results from parametric ANOVA anaysis, pooled T-tests were used for pairwise comparisons. Non-significant results from a non-parametric ANOVA analysis , separate variance T Tests for pairwise comparison were used.
The Kruskal-Wallis test, followed by the Mann-Whitney U test , where appropriate was used to statistically evaluate non-parametric data. Incidence data were compared using the Fisher's extract test. With the exception of frequency data for fetal malformations and variations, statistical analyses were performed using BMDP statistical software. For all statistical tests, the critical level of significance was set at 0.05 (two tailed).
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: changes in weight gain, skin irritation, changes in liver weight, changes in blood parameters

Details on maternal toxic effects:
Rat dams administered 150 mg/kg/day DEA showed significant decreases in body weight gains at the end of the dosing period and during the post dosing period (GD 15-21). When corrected for gravid uterine weight, this corresponded to 4.5% decrease in body weight which is significantly less than body weights in control animals. At 150 and 500 mg/kg bw/day , there was no treatment related declines in maternal weight gain at any interval. No effect on food consumption was observed at any dose level. Following administration of DEA rats administered 150 and 1500 mg/kg bw/day had a significant increase in weight gain, although there was no evidence to show a clear dose response relationship.
Rats in the 500 and 1500 mg/kg bw/day group showed clinical signs of skin irritation which were dose dependent in incidence and severity. This condition persisted into the post treatment observation period in the high dose group. No significant effects on skin irritation were observed in the control group of 50 mg/kg bw/day group.
Analysis of blood samples collected at the time of necropsy showed that DEA slightly reduced red blood cell parameters , including haematocrit, MCV, MCH, haemoglobin concentration and erythrocyte number at all concentrations tested. Rats in th 1500 mg/kg bw/day group had increased numbers of leukocytes and lymphocytes, but decreased platelet numbers. Changes in red cell morphology (poikilocytosis, anisocytosis, polychromasia) were observed in all dose groups.
No changes in absolute or relative liver weights were observed in any dose group, but significant dose dependent increases in both absolute and relative kidney weights were observed at the 500 and 1500 mg/kg bw/day dose groups.
DEA had no effect on pregnancy rate, corpora lutea number, implantation number, litter size, resorption rate, number of dead foetuses, foetal body weight, foetal sex ratio or gravid uterine weight ant any dose level tested.
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
380 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: Minor ossification effects that occurred only with significant maternal toxicity

Details on embryotoxic / teratogenic effects:
Dermal administration of DEA had no effect on the overall incidence of external, visceral or skeletal malformations or variations observed in rat foetuses. Litters fromthe 1500 mg/kg bw/day group had statistically significant increased incidences of six skeletal alterations poorly ossified cervical centrum, thoracic centrum , parietal, hindlimbs and forelimbs.The only statistical significant variation in the skull ar 1500 mg/kg was poorly ossified parietal. However, ossification patterns in adjacent bones suggest a general minor delay in ossification of the skull, There was no effect on any of these developmental parameters at 500 mg/kg bw/day.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Dermally applied DEA did not produce any teratogenicity effects, even at doses which cause severe skin irritation. Increased incidences of six variation were seen at the high dose group. However, these effects were considered to involve minor delays in ossification which are thought to be reversible and not to have a long lasting effect on the health or survival of the offspring. A NOEL could not be identified for maternal toxicity due to haematological effects even at the low dose group. The maternal LOAEL was considered to be 150 mg/kg bw/day. The NOAEL for developemntal toxicity was considered to be 380 mg/kg bw/day instead of 500 mg/kg bw/day due to a dosing deficit on GD 12-15.
Executive summary:

Female CD rats were dermally exposed to DEA at concentrations of 0, 150, 500 or 1500 mg/kg bw/day from GD 6 to 15. Maternal toxicity included a 4.5 % decrease in body weight in animals in the 150 mg/kg bw/day group when compared with control animals, along with clinical signs of skin irritation in the mid and high dose group which were dose dependent in incidence and severity. Analysis of maternal blood samples collected at time of necropsy showed that DEA slightly reduced red blood cell parameters including haematocrit, MCV, MCH, haemoglobin concentrations and erythrocyte numbers in all dose groups. No changes in absolute or relative liver weights were observed in any dose group, but significant dose dependent increases in both absolute and relative kidney weights were observed at the 500 and 1500 mg/kg bw/day dose groups. DEA had no effect on pregnancy rate, corpora lutea number, implantation number, litter size, resorption rate, number of dead foetuses, foetal body weight, foetal sex ratio or gravid uterine weight at any dose level tested.

Repeated dermal administration of DEA had no effect on the overall incidence of external, visceral or skeletal malformations.Litters from the 1500 mg/kg bw/day group had statistically significant increased incidences of six skeletal alterations poorly ossified cervical centrum, thoracic centrum, and parietal, hind limbs and fore limbs. The only statistical significant variation in the skull at 1500 mg/kg was poorly ossified parietal. However, ossification patterns in adjacent bones suggest a general minor delay in ossification of the skull. There was no effect on any of these developmental parameters at 500 mg/kg bw/day.

Therefore, in conclusion dermally applied DEA did not produce any teratogenicity effects, even at doses which cause severe skin irritation. Increased incidences of six skeletal variations were seen at the high dose group. However, these effects were considered to involve minor delays in ossification which are thought to be reversible and not to have a long lasting effect on the health or survival of the offspring. A NOEL could not be identified for maternal toxicity due to haematological effects even at the low dose group. The maternal LOAEL was considered to be 150 mg/kg bw/day. The NOAEL for developmental toxicity was considered to be 380 mg/kg bw/day instead of 500 mg/kg bw/day due to a dosing deficit on GD 12-15.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Both available studies were conducted in accordance with the standardised guideline OECD 422 under GLP conditions and were assigned a reliability score of 2 in accordance with the criteria set forth by Klimisch (1997).
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
200 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study was conducted on the read across material diethanolamine in accordance with the standardised guideline OECD 414 under GLP conditions. It was assigned a reliability score of 2 in accordance with the criteria set forth by Klimisch (1997).
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
380 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
There is a dermal pre-natal study available in rats and in rabbits on DEA, one of the two source chemicals. These studies were assigned a Klimisch = 2 as the data is based on read across.
Additional information

No reproductive or developmental studies are available on the target substance. However, according the TIMES rat liver S9 metabolism simulator, the target substance is fully metabolized into tall oil and diethanolamine (DEA). Therefore, data on these two source substances are used to fulfill the reproductive endpoints. Further discussion on the metabolism of the target substance is given in the Read Across Justification Document in Section 13.

Effect on developmental toxicity: via oral route

The first combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the read across material tall oil 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, small decreases were noted in ovary weight in females; 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. No effects were seen in offspring.

Under the conditions of this study, no effects were observed on offspring, therefore the NOEL for developmental parameters was considered to be 20 000 ppm, equivalent to 2000 mg/kg bw/day.

 

The second combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the read across material tall oil, compound with ethanolamine in accordance with the standardised guideline OECD 422 under GLP conditions.

Three groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats were dosed with the vehicle (UHP water) following the same dosing regimen as the treated animals and were used as controls. The males were dosed for 14 days prior to pairing, during pairing and until the day prior to necropsy. The females were dosed for 14 days prior to pairing, during pairing and gestation, and up to and including Day 3 of lactation.

There were no unscheduled deaths on the study and administration of the test material was well tolerated; there was no effect of treatment on fertility or mating performance. There was no effect of treatment with the test material on the mean duration of gestation. The mean number of pups born per litter, sex ratio and mean live birth, viability and cumulative survival indices in all treated groups were comparable with controls. There were no unscheduled pup deaths on the study.

Group mean pup body weight gains between Days 1 and 4 of age were unaffected by maternal treatment with the test material and there were no necropsy findings for pups considered to be related to maternal treatment with the test material.

Under the conditions of this study, in the Wistar strain rat, the No Observed Adverse Effect Level (NOAEL) for developmental effects was considered to be 1000 mg/kg/day.

 

Effect on developmental toxicity: via inhalation route

The potential of the read across material diethanolamine to cause developmental effects was investigated in accordance with the standardised guideline OECD 414 under GLP conditions.

Female Wistar rats were exposed to the test material at concentrations of 0.01, 0.05 and 0.2 mg/L (25 animals per dose level) from day 6 of gestation to day 15.

Under the conditions of this study, the NOAEC for teratogenicity was determined to be ≥0.2 mg/L air, equivalent to 200 mg/m³, whilst the NOAEC for maternal toxicity was determined to be 0.05 mg/L air.

Effect on developmental toxicity:via dermal route

Female CD rats were dermally exposed to DEA at concentrations of 0, 150, 500 or 1500 mg/kg bw/day from GD 6 to 15. Maternal toxicity included a 4.5 % decrease in body weight in animals in the 150 mg/kg bw/day group when compared with control animals, along with clinical signs of skin irritation in the mid and high dose group which were dose dependent in incidence and severity. Analysis of maternal blood samples collected at time of necropsy showed that DEA slightly reduced red blood cell parameters including haematocrit, MCV, MCH, haemoglobin concentrations and erythrocyte numbers in all dose groups. No changes in absolute or relative liver weights were observed in any dose group, but significant dose dependent increases in both absolute and relative kidney weights were observed at the 500 and 1500 mg/kg bw/day dose groups. DEA had no effect on pregnancy rate, corpora lutea number, implantation number, litter size, resorption rate, number of dead foetuses, foetal body weight, foetal sex ratio or gravid uterine weight at any dose level tested.

Repeated dermal administration of DEA had no effect on the overall incidence of external, visceral or skeletal malformations.Litters from the 1500 mg/kg bw/day group had statistically significant increased incidences of six skeletal alterations poorly ossified cervical centrum, thoracic centrum, and parietal, hind limbs and fore limbs. The only statistical significant variation in the skull at 1500 mg/kg was poorly ossified parietal. However, ossification patterns in adjacent bones suggest a general minor delay in ossification of the skull. There was no effect on any of these developmental parameters at 500 mg/kg bw/day.

Therefore, in conclusion dermally applied DEA did not produce any teratogenicity effects, even at doses which cause severe skin irritation. Increased incidences of six skeletal variations were seen at the high dose group. However, these effects were considered to involve minor delays in ossification which are thought to be reversible and not to have a long lasting effect on the health or survival of the offspring. A NOEL could not be identified for maternal toxicity due to haematological effects even at the low dose group. The maternal LOAEL was considered to be 150 mg/kg bw/day. The NOAEL for developmental toxicity was considered to be 380 mg/kg bw/day instead of 500 mg/kg bw/day due to a dosing deficit on GD 12-15.

In a study similar to OECD 414, DEA was dermally exposed to pregnant rabbits from gestation day 6 to 18 at concentrations of 0, 35, 100 or 350 mg/kg bw/day. Severe skin irritation, including erythema, oedema and necrosis was seen at the dosing site of animals exposed to 350 mg/kg bw/day. No skin lesions were observed in any other dosing group. Overall, there was a decrease in gestational body weight gain in the mid and high dose group during the treatment period, although this difference did not reach statistical significance. Rabbits in the low dose group had a slight increase in maternal body weight and bod weight gain compared to controls, although this finding was considered to be incidental and non-treatment related. Colour changes in the kidneys were observed in approximately 50% of treated dams in the high dose group, compared with 17% in control animals. Although not statistically significant, absolute and relative liver weights and relative kidney weights were increased by 10,16 and 7%, respectively, in the 350 mg/kg bw/day group. There was a significant decrease in the litter incidence of visceral variations at 35 mg/kg bw/day. This is thought to be an incidental finding as there was no dose response. One foetus from a dam in the high dose group showed a greater variety of malformations than any other foetus including ovoid lenses, common trucus, ventricular septal defect, herniated diaphragm, extra lumbar centrum No. 8, extra bilateral lumbar arch No. 8, bone island at caudal segment No. 2 and duplicated, mis-shaped, and fused sternebra. The overall incidence of malformations in the high dose DEA group ( 4.5% foetuses, 30.8% of litters) was similar to the incidence seen in the control animals (6.7% foetuses, 25.0% of litters.) There were no other note-worthy developmental effects seen. Therefore, the NOEL for maternal toxicity was considered to be 35 mg/kg bw/day based on decreased body weight at all other dose levels. The NOEL for developmental toxicity was considered to be 350 mg/kg bw/day.

 

 


Justification for selection of Effect on developmental toxicity: via oral route:
This endpoint was addressed on a weight of evidence basis with two combined repeated dose toxicity study with reproduction/developmental toxicity screening studies being carried out on the read across materials tall oil and tall oil, compound with ethanolamine.
The study conducted on tall oil, compound with ethanolamine was selected as key as a precaution on the basis that the NOAEL achieved in this study was lower than the NOEL reported for the study on tall oil.
It is considered that these studies together are an accurate reflection of the total composition of the registered substance, Tall Oil, compound with diethanolamine.
Therefore, the extended one generation reproductive toxicity study is not scientifically justified and is being waived.

Justification for selection of Effect on developmental toxicity: via inhalation route:
Only one study available.

Justification for selection of Effect on developmental toxicity: via dermal route:
The key study was conducted on the read across material diethanolamine in accordance with the standardised guideline OECD 414 under GLP conditions. It was assigned a reliability score of 2 in accordance with the criteria set forth by Klimisch (1997).

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for toxicity to reproduction.

No reproductive or developmental studies are available on the target substance. However, according the TIMES rat liver S9 metabolism simulator, the target substance is fully metabolized into tall oil and diethanolamine (DEA). Therefore, data on these two source substances are used to fulfill the reproductive endpoints. Further discussion on the metabolism of the target substance is given in the Read Across Justification Document in Section 13.