Tall oil

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21st March 2002 to 26th February 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

It was based on the version of the test guideline available at the time (Adopted 22nd March 1996). There are some differences in the information reported compared with stipulations in the current version of OECD 422 (Adopted 29th July 2016). These are: • Functional Observations not performed • Thyroid hormones not measured • Study terminated on PND6, rather than continuing until minimally PND13 • Anogenital distance was not reported There were no deviations from the study plan that impacted upon the integrity of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

no certificate of analysis or details of test substance supplied, however given the nature of the distillation products this deviation was not thought to have affected the integrity of the study.

Principles of method if other than guideline:

N/A

GLP compliance:

yes

Limit test:

no

Justification for study design:

The dose levels were selected and agreed with the Sponsor, following evaluation of existing toxicological data. This included data from a one-week dose range finding study in rats carried out under a separate contract and project number at Inveresk (Inveresk Project No. 493160).

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Tall Oil
- Substance type: Complex mixture
- Physical state: dark brown liquid
- Analytical purity: No data
- Composition of test material, percentage of components: Not supplied
- Purity test date: No data
- Lot/batch No.: 7252-30
- Expiration date of the lot/batch: 15.05.05
- Stability under test conditions: No data
- Storage condition of test material: Room temperature, dark, under nitrogen

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
At room temperature, in the dark, under nitrogen>

STABILITY UNDER TEST CONDITIONS
Not evaluated during study.

FORM AS APPLIED IN THE TEST
Batches of diet were prepared weekly.
An appropriate quantity of the test item was dissolved in a suitable volume of acetone. This solution was added to a suitable quantity of untreated diet, then mixed for ca one hour with fan assisted venting to remove the ethanol 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 ca 20 min. 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.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat is a standard rodent species for the reproduction toxicity testing in animals required by regulatory authorities. The normal processes of reproduction in the rat are well documented in the test laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: Males: 180-190 g. Females: 113-161 g.
- Fasting period before study: No
- Housing: Initially two per polypropylene cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ±2
- Humidity (%): 50 ±15
- Air changes (per hr): minimum 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 08.04.02 To: 27.05.02

Administration / exposure

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

The test material was administered orally because this is a possible route of exposure in humans. Administration via the diet is an established experimental routine in rats.

The dose levels were selected and agreed with the Sponsor, following evaluation of existing toxicological data. This included data from a one-week dose range finding study in rats carried out under a separate contract and project number at Inveresk (Inveresk Project No. 493160).

Details on 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 the formulated diets was undertaken regarding concentration and homogeneity. Diet prepared for Week 1 and Week 4 of treatment was sampled. Triplicate samples of each formulation, including control, were taken immediately after preparation. The samples were analysed by the Inveresk Product Chemistry Laboratory using a method previously validated in the Inveresk laboratory under a separate protocol and contract (Inveresk Project No. 491836).

Duration of treatment / exposure:

The males were treated for at least four weeks overall, starting from two weeks prior to mating until termination. The females were treated for two weeks prior to mating, then through mating, until termination after Day 4 of lactation.

Frequency of treatment:

Continuous in diet

Details on study schedule:

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

Group 1 (control)

Doses / concentrations 2

Dose / conc.:

1 000 ppm (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

5 000 ppm (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

20 000 ppm (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

Ten

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected and agreed following evaluation of existing data and a one week dose range-finding study in rats.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No satellite groups.

Positive control:

No positive control

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily


BODY WEIGHT: Yes
- Time schedule for examinations: Males: once during the week prior to the commencement of dosing and once weekly thereafter. Females: once during the week prior to commencement of treatment, and weekly thereafter until the start of the mating period, 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 is the day of parturition).


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Group mean achieved dosages of test substance calculated: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 5 of dosing for males and on Day 6 of lactation for females.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: Five males and five females
- Parameters checked in table No.1 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 5 of dosing for males and on Day 6 of lactation for females.
- Animals fasted: No
- How many animals: Five males and five females
- Parameters checked in table No.1 were examined.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

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


GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following four weeks of treatment.
- Maternal animals: All surviving animals after four days of lactation.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 2 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- All of the F1 offspring.
- These animals were subjected to postmortem examinations: externally visible abnormalities only.


GROSS NECROPSY: None


HISTOPATHOLOGY / ORGAN WEIGTHS: None

Statistics:

Body weight 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 likewise, and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. Histology incidence data were analysed using Fisher's Exact Probability Test. The following pairwise comparisons were performed against the Control group (Group 1): Control group vs Low dose; control group vs intermediate dose; Control group vs high dose. All statistical tests were two-sided and performed at the 5% significance level.

Reproductive indices:

Fertility Index (female) = Number Pregnant/Number Paired
Fertility Index (male) = Number Siring a Litter/Number paired
Gestation Index = Number bearing live pups/Number pregnant
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)

Offspring viability indices:

Viability Index = Number of pups live on Day 4 of lactation/number live on Day 0

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

All clinical observations were considered to be consistent with those normally seen in rats of this age and strain.

Mortality:

no mortality observed

Description (incidence):

N/A

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Please see Table 3-Table 5 for data.

At 20000 ppm, there was a transient decrease in weight gain in both sexes. In males, decreased weight gain was most notable for 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 Day 7- 20 of gestation, compared to the Control animals.

There were no obvious effects of treatment at 5000 or 1000 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

FOOD CONSUMPTION

Please see Table 6 to Table 8 for data.

At 20000 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.

COMPOUND INTAKE

Please see Table 9 for data.

Food efficiency:

not examined

Description (incidence and severity):

N/A

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

not examined

Description (incidence and severity):

N/A

Ophthalmological findings:

not examined

Description (incidence and severity):

N/A

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Please see Table 10 & Table 11 for data.

At 20000 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 biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Please see Table 12 & Table 13 for data.

Alkaline phosphatase levels were significantly Increased in females at 5000 and 20000 ppm, and in males at 20000 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 sjze, it was considered that the difference was too small to reflect an effect of treatment.

Total bilirubin was increased in both sexes at 20000 ppm.

In addition, at 20000 ppm, cholesterol levels were Increased in males; albumin (and consequently total protein) were reduced in females.

Endocrine findings:

not examined

Description (incidence and severity):

N/A

Urinalysis findings:

not examined

Description (incidence and severity):

N/A

Behaviour (functional findings):

not examined

Description (incidence and severity):

N/A

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

N/A

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All histology findings were typical of spontaneously arising background findings in rats of this strain and age.

Other effects:

not examined

Description (incidence and severity):

N/A

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

N/A

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

N/A

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Please see Table 16 to Table 18 for data.

Mating performance was not affected by treatment. There were no obvious effects on the duration of gestation at any of the dose levels applied.

At 20000 ppm mean number of implant sites per pregnancy was marginally decreased and hence 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.

Details on results (P0)

N/A

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

ca. 1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Decreased weight gain and food consumption in both sexes at 20000 ppm. Changes in liver function in both sexes at 20000 ppm. At 5000 ppm there was increased liver weight and alkaline phosphatase in both sexes.

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Description (incidence and severity):

N/A

Mortality / viability:

not specified

Description (incidence and severity):

N/A

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Please see Table 19 for data.

At 20000 ppm, mean litter weights were slightly reduced compared to the Controls, reflecting the decrease In litter size.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

N/A

Food efficiency:

not examined

Description (incidence and severity):

N/A

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

not examined

Description (incidence and severity):

N/A

Ophthalmological findings:

not examined

Description (incidence and severity):

N/A

Haematological findings:

not examined

Description (incidence and severity):

N/A

Clinical biochemistry findings:

not examined

Description (incidence and severity):

N/A

Urinalysis findings:

not examined

Description (incidence and severity):

N/A

Sexual maturation:

not examined

Description (incidence and severity):

N/A

Anogenital distance (AGD):

not examined

Description (incidence and severity):

N/A

Nipple retention in male pups:

not examined

Description (incidence and severity):

N/A

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

N/A

Gross pathological findings:

no effects observed

Description (incidence and severity):

N/A

Histopathological findings:

not examined

Description (incidence and severity):

N/A

Other effects:

not examined

Description (incidence and severity):

N/A

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Description (incidence and severity):

N/A

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Description (incidence and severity):

N/A

Details on results (F1)

N/A

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 20 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 3                Body Weights & Body Weight Gain (g). Male

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
BODY WEIGHT
DAY 0	MEAN SD N	320 17 10	313 9 10	316 11 10	313 10 10
DAY 7	MEAN SD N	374 26 10	369 16 10	373 16 10	351** 13 10
DAY 14	MEAN SD N	414 34 10	408 17 10	415 20 10	386* 18 10
DAY 21	MEAN SD N	447 37 10	437 19 10	444 26 10	413** 22 10
DAY 28	MEAN SD N	468 45 10	460 23 10	471 28 10	434 29 10
BODY WEIGHT GAIN
DAY 0 to DAY 28	MEAN SD N	149 30 10	147 16 10	154 20 10	121* 24 10

Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001

 

Table 4                Body Weights & Body Weight Gain (g). Female – Pre-mating

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
BODY WEIGHT
DAY 0	MEAN SD N	194 9 10	199 15 10	197 8 10	188 9 10
DAY 7	MEAN SD N	217 13 10	221 18 10	219 10 10	200** 9 10
DAY 14	MEAN SD N	237 16 10	240 22 10	237 10 10	214** 15 10
BODY WEIGHT GAIN
DAY 0 to DAY 14	MEAN SD N	43 9 10	41 12 10	40 4 10	25** 12 10

Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001

 

Table 5                Mean Body Weights & Mean Body Weight Gain (g). Female – Gestation and Lactation

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
BODY WEIGHT
DAY 0 Of gestation	MEAN	245	247	240	218
DAY 7 Of gestation	MEAN	286	285	275	256
DAY 14 Of gestation	MEAN	329	320	317	288
DAY 20 Of gestation	MEAN	410	407	388	345
BODY WEIGHT GAIN
DAY 0 to DAY 20	MEAN (% of Control)	165 -	160 97	148 90	127 77
BODY WEIGHT
DAY 1 Of lactation	MEAN	298	286	273	261
DAY 4 Of Lactation	MEAN	319	313	300	281

 

 

Table 6                Food Consumption (g/animal/day). Male

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
DAYS 0-7	MEAN SD N (CAGE)	31.5 2.2 5	30.9 2.0 5	32.6 1.6 5	27.2** 2.2 5
DAYS 7-14	MEAN SD N (CAGE)	35.5 3.9 5	34.1 1.9 5	34.9 1.3 5	32.1 2.4 5
DAYS 21-28	MEAN SD N (CAGE)	32.0 2.8 5	31.5 1.5 5	32.4 2.5 5	29.9 1.9 5

Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001

 

Table 7                Food Consumption (g/animal/day). Female – pre-mating

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
DAYS 0-7	MEAN SD N (CAGE)	21.1 1.0 5	21.7 1.2 5	20.5 2.0 5	17.2*** 1.3 5
DAYS 7-14	MEAN SD N (CAGE)	22.9 1.3 5	22.6 1.1 5	21.6 1.5 5	20.3** 1.5 5

Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001

 

Table 8                Mean Food Consumption (g/animal/day). Female – Gestation and Lactation

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
DAYS 0-7 Of gestation	MEAN	26.9	26.5	26.2	23.9
DAYS 7-14 Of gestation	MEAN	29.9	30.0	27.9	26.7
DAYS 14-20 Of gestation	MEAN	31.8	32.8	32.6	30.1
DAYS 0-4 Of lactation	MEAN	33.9	31.9	31.6	30.1

 

Table 9                Group Mean Achieved Dosage of Test Item (mg/kg/day)

	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
MALES
WEEK 1	91	473	1639
WEEK 2	88	443	1742
WEEK 4	70	354	1412
FEMALES
WEEK 1	103	493	1773
WEEK 2	98	474	1961
DAYS 0-7 (OF GESTATION)	100	509	2017
DAYS 7-14 (OF GESTATION)	99	471	1963
DAYS 14-20 (OF GESTATION	90	462	1902
DAYS 1-4 (OF LACTATION)	107	551	2221

 

Table 10             Haematology, Week 5. Males

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
Hb	MEAN SD N	16.1 0.6 5	16.1 0.4 5	15.5 0.1 5	15.7 0.5 5
RBC	MEAN SD N	8.28 0.32 5	8.34 0.23 5	7.95 0.27 5	8.10 0.25 5
Hct	MEAN SD N	0.453 0.019 5	0.452 0.014 5	0.442 0.004 5	0.440 0.015 5
MCH	MEAN SD N	19.5 0.4 5	19.3 0.5 5	19.5 0.7 5	19.4 0.3 5
MCV	MEAN SD N	54.7 1.5 5	54.2 1.4 5	55.5 1.7 5	54.3 0.9 5
MCHC	MEAN SD N	35.6 0.3 5	35.7 0.4 5	35.1* 0.2 5	35.7 0.4 5
WBC	MEAN SD N	14.50 2.21 5	14.33 2.85 5	15.82 4.05 5	12.97 3.93 5
Neut	MEAN SD N	1.54 0.37 5	2.30 1.46 5	1.57 0.49 5	1.49 0.74 5
Lymp	MEAN SD N	12.39 1.96 5	11.41 3.59 5	13.71 4.23 5	10.91 3.02 5
Mono	MEAN SD N	0.25 0.07 5	0.28 0.10 5	0.20 0.02 5	0.22 0.11 5
Eos	MEAN SD N	0.19 0.05 5	0.17 0.07 5	0.16 0.07 5	0.15 0.10 5
Baso	MEAN SD N	0.04 0.02 5	0.04 0.02 5	0.06 0.03 5	0.04 0.02 5
LUC	MEAN SD N	0.09 0.03 5	0.12 0.03 5	0.12 0.05 5	0.16 0.10 5
Plat	MEAN SD N	1023 102 5	975 122 5	967 119 5	994 195 5
PT	MEAN SD N	13 1 5	13 1 5	13 1 5	12* 1 5

Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001

 

 

Table 11             Haematology, Week 7. Females

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
Hb	MEAN SD N	14.0 1.0 5	13.9 0.6 5	13.2 0.4 5	13.0 1.7 5
RBC	MEAN SD N	7.30 0.46 5	7.13 0.31 5	6.88 0.29 5	6.87 0.85 5
Hct	MEAN SD N	0.394 0.029 5	0.390 0.021 5	0.370 0.014 5	0.365 0.048 5
MCH	MEAN SD N	19.1 0.6 5	19.5 0.2 5	19.2 0.4 5	19.0 0.4 5
MCV	MEAN SD N	54.0 1.4 5	54.7 1.3 5	53.8 1.3 5	53.2 1.6 5
MCHC	MEAN SD N	35.4 0.6 5	35.7 0.7 5	35.7 0.4 5	35.7 0.3 5
WBC	MEAN SD N	12.99 3.46 5	12.79 3.85 5	12.15 2.84 5	8.89 2.28 5
Neut	MEAN SD N	3.84 1.68 5	3.30 1.19 5	3.62 1.09 5	2.44 0.98 5
Lymp	MEAN SD N	8.55 2.89 5	8.78 2.85 5	7.99 2.05 5	6.04 1.83 5
Mono	MEAN SD N	0.28 0.13 5	0.34 0.22 5	0.26 0.04 5	0.15* 0.04 5
Eos	MEAN SD N	0.18 0.07 5	0.15 0.07 5	0.14 0.03 5	0.17 0.17 5
Baso	MEAN SD N	0.03 0.03 5	0.04 0.03 5	0.03 0.01 5	0.02 0.01 5
LUC	MEAN SD N	0.11 0.06 5	0.17 0.10 5	0.12 0.07 5	0.08 0.02 5
Plat	MEAN SD N	1073 133 5	1061 145 5	1110 170 5	906 478 5
PT	MEAN SD N	14 1 5	14 1 4	14 1 4	13 2 3

Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001

 

Table 12             Clinical Chemistry, Week 5. Male

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
Urea	MEAN SD N	7.3 0.5 5	7.4 1.1 5	6.5 0.5 5	6.4 0.6 5
Glu	MEAN SD N	7.92 0.42 5	7.95 0.76 5	8.12 0.88 5	7.90 0.29 5
AST	MEAN SD N	86 8 5	92 15 5	92 6 5	83 5 5
ALT	MEAN SD N	59 2 5	71 8 5	70 11 5	65 9 5
AP	MEAN SD N	683 39 5	699 137 5	842 183 5	1891** 905 5
Na	MEAN SD N	144 1 5	144 2 5	143 2 5	142 3 5
k	MEAN SD N	4.9 0.2 5	5.0 0.1 5	4.9 0.3 5	5.0 0.2 5
Cl	MEAN SD N	101 1 5	102 2 5	101 2 5	101 4 5
TP	MEAN SD N	68 2 5	70 2 5	66 2 5	66 2 5
Alb	MEAN SD N	41 1 5	43 1 5	41 1 5	41 2 5
AG-R	MEAN SD N	1.6 0.2 5	1.6 0.1 5	1.6 0.3 5	1.7 0.1 5
Chol	MEAN SD N	2.0 0.4 5	1.9 0.3 5	2.0 0.3 5	2.4* 0.1 5
Crea	MEAN SD N	51 2 5	53 2 5	52 3 5	56 4 5
Ca	MEAN SD N	2.91 0.06 5	2.95 0.07 5	2.94 0.07 5	2.94 0.07 5
Phos	MEAN SD N	2.01 1.13 5	2.58 0.09 5	2.60 0.14 5	2.08 1.17 5
T. Bi	MEAN SD N	0.6 0.2 5	1.0 0.6 5	0.9 0.2 5	1.7*** 0.4 5

Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001

 

Table 13             Clinical Chemistry, Week 7. Female

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
Urea	MEAN SD N	10.7 1.2 5	11.1 2.7 5	11.8 1.2 5	10.5 0.7 5
Glu	MEAN SD N	6.68 0.90 5	6.65 0.70 5	6.00 0.35 5	6.44 0.77 5
AST	MEAN SD N	96 17 5	88 17 5	98 8 5	84 14 5
ALT	MEAN SD N	105 23 5	114 31 5	128 28 5	113 21 5
AP	MEAN SD N	509 203 5	638 152 5	980* 378 5	1649*** 929 5
Na	MEAN SD N	145 3 5	141 3 5	144 1 5	142 3 5
k	MEAN SD N	5.3 0.7 5	5.6 0.3 5	5.9 0.4 5	5.9 0.5 5
Cl	MEAN SD N	106 4 5	103 4 5	105 2 5	104 1 5
TP	MEAN SD N	64 1 5	65 4 5	62 2 5	59* 4 5
Alb	MEAN SD N	40 1 5	39 2 5	39 2 5	35*** 1 5
AG-R	MEAN SD N	1.6 0.1 5	1.5 0.1 5	1.7 0.2 5	1.4* 0.1 5
Chol	MEAN SD N	2.4 0.5 5	2.2 0.5 5	1.9* 0.1 5	2.7 0.3 5
Crea	MEAN SD N	59 4 5	58 2 5	62 4 5	63 3 5
Ca	MEAN SD N	2.84 0.12 5	2.90 0.06 5	2.79 0.08 5	2.77 0.09 5
Phos	MEAN SD N	1.89 0.27 5	1.93 0.31 5	1.58 0.21 5	1.86 0.24 5
T. Bi	MEAN SD N	0.9 0.2 5	0.9 0.6 5	1.4 0.6 5	1.9** 0.5 5

Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001

 

Table 14             Absolute Organ Weights (Following Covariance Analysis). Males

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
BODY WEIGHT	MEAN SE N	458 11 10	458 11 10	458 11 10	458 11 10
ADRENAL GLANDS	MEAN SE N	0.0757 0.0036 10	0.0803 0.0036 10	0.0674 0.0036 10	0.0634* 0.0038 10
BRAIN	MEAN SE N	2.10 0.03 10	2.12 0.03 10	2.12 0.03 10	2.08 0.03 10
EPIDIDYMIDES	MEAN SE N	1.2041 0.0292 10	1.2032 0.0287 10	1.2048 0.0291 10	1.2047 0.0311 10
HEART	MEAN SE N	1.72 0.06 10	1.82 0.06 10	1.73 0.06 10	1.72 0.06 10
KIDNEYS	MEAN SE N	3.77 0.10 10	3.85 0.10 10	4.01 0.10 10	4.07 0.11 10
LIVER	MEAN SE N	17.88 0.53 10	18.88 0.52 10	19.46* 0.53 10	20.12** 0.56 10
LUNG	MEAN SE N	1.82 0.06 9	1.86 0.05 10	1.76 0.05 10	1.75 0.06 10
PITUITARY GLAND	MEAN SE N	0.0013 0.0001 10	0.012 0.001 10	0.013 0.001 10	0.011 0.001 10
PROSTATE	MEAN SE N	0.771 0.046 10	0.777 0.045 10	0.704 0.046 10	0.761 0.049 10
SPLEEN	MEAN SE N	0.85 0.03 10	0.83 0.03 10	0.85 0.03 10	1.04*** 0.03 10
SALIVARY GLANDS	MEAN SE N	0.7770 0.0240 10	0.7572 0.0236 10	0.7501 0.0239 10	0.7978 0.0255 10
TESTES	MEAN SE N	3.55 0.08 10	3.51 0.07 10	3.69 0.08 10	3.74 0.08 10
THYMUS	MEAN SE N	0.491 0.032 10	0.415 0.031 10	0.435 0.032 10	0.385 0.034 10
THYROID GLANDS	MEAN SE N	0.0219 0.0010 10	0.0233 0.0009 10	0.0225 0.0010 9	0.0230 0.0010 10

Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001

 

Table 15             Absolute Organ Weights (Following Covariance Analysis). Females

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
BODY WEIGHT	MEAN SE N	310 7 10	310 7 10	310 7 10	310 8 8
ADRENAL GLANDS	MEAN SE N	0.0911 0.0037 10	0.0843 0.0032 10	0.0791 0.0032 10	0.0774 0.0042 8
BRAIN	MEAN SE N	1.87 0.02 10	1.87 0.02 10	1.90 0.02 10	1.96 0.03 8
HEART	MEAN SE N	1.12 0.04 10	1.16 0.04 10	1.25 0.04 10	1.18 0.05 8
KIDNEYS	MEAN SE N	2.59 0.05 10	2.53 0.05 10	2.47 0.05 10	2.53 0.06 8
LIVER	MEAN SE N	16.69 0.48 10	16.16 0.43 10	17.77 0.43 10	17.87 0.55 8
LUNG	MEAN SE N	1.32 0.04 9	1.38 0.03 10	1.29 0.03 10	1.37 0.04 8
OVARIES	MEAN SE N	0.114 0.005 10	0.106 0.004 10	0.104 0.004 10	0.097 0.006 8
PITUITARY GLAND	MEAN SE N	0.014 0.001 10	0.016 0.001 10	0.015 0.001 10	0.013 0.001 8
SPLEEN	MEAN SE N	0.59 0.03 10	0.63 0.03 10	0.68 0.03 10	0.72 0.03 8
SALIVARY GLANDS	MEAN SE N	0.6038 0.0208 10	0.6027 0.0184 10	0.6121 0.0185 10	0.5755 0.0238 8
THYMUS	MEAN SE N	0.198 0.024 10	0.233 0.021 10	0.201 0.021 10	0.242 0.028 8
THYROID GLANDS	MEAN SE N	0.0166 0.0010 9	0.0165 0.0009 10	0.0152 0.0009 10	0.0171 0.0011 8
UTERUS	MEAN SE N	0.53 0.02 10	0.53 0.02 10	0.51 0.02 10	0.50 0.03 8

Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001

 

Table 16             Mating performance and fertility indices

	GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
Number of males paired	10	10	10	10
Number of siring males	10	10	10	8
Number of females paired	10	10	10	10
Pregnant females	10	10	10	8
Number of females producing a liver litter	10	10	10	8
Fertility Index, males (%)	100	100	100	80
Fertility Index, females (%)	100	100	100	80
Gestation Index (%)	100	100	100	100

 

 

Table 17             Implantation sites summary

		GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
IMPLANTATIONS	MEAN SD N	13.4 3.6 10	15.4 2.5 10	14.2 3.8 10	12.1 1.5 10

 

 

Table 18             Developmental data

	GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
Mean number of live pups per litter ± SD, Day 0	12.4 ± 3.5	14.7 ± 2.5	13.1 ± 3.4	11.4 ± 1.7
Mean number of live pups per litter ± SD, Day 1	12.3 ± 3.3	14.0 ± 3.9	13.0 ± 3.4	11.4 ± 1.7
Mean number of live pups per litter ± SD, Day 4	12.3 ± 3.3	13.1 ± 4.7	13.0 ± 3.4	11.4 ± 1.7
Birth index (%)	90	96	93	97
Live birth index (%)	99	100	100	97
Viability index (%)	99	88	99	100

 

 

Table 19             Group Mean Litter and Pup Weight (g) ± SD

 

	GROUP 1 CONTROL	GROUP 2 1000 PPM	GROUP 3 5000 PPM	GROUP 4 20000 PPM
LITTER
Day 1	84 ± 17	85 ± 21	82 ± 16	79 ± 9
Day 4	126 ± 23	124 ± 50	121 ± 22	117 ± 12
Mean of Litter Mean Pup Weight
MALES
Day 1	7.2 ± 0.9	6.4 ± 0.9	6.7 ± 1.2	7.2 ± 0.8
Day 4	10.8 ± 2.0	9.7 ± 1.9	10.1 ± 2.2	10.7 ± 1.4
FEMALES
Day 1	6.9 ± 1.2	5.9 ± 0.8	6.2 ± 1.0	6.8 ± 0.6
Day 4	10.5 ± 2.2	9.0 ± 2.6	9.4 ± 1.7	10.2 ± 1.2

 

Applicant's summary and conclusion

Conclusions:

In a good quality Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted to OECD test guideline 422, and GLP, the parental NOAEL, for Tall Oil administered continuously in the diet to rats (Sprague-Dawley), was 1000ppm. The NOAEL for reproductive toxicity was 5000 ppm.

Executive summary:

Four groups of 10 male and 10 female Sprague-Dawley rats received Tall Oil in diets at concentrations of 0, 1000, 5000 and 20000 ppm. The males were dosed for at least four weeks, starting from two weeks prior to mating. The females were dosed from two weeks prior to mating until at least Day 6 of lactation. The animals were monitored for clinical signs, body weight, food consumption, mating and litter performance. Blood samples were taken from five males (during week five) and five females (lactation Day 6) per group for laboratory investigations. All parental animals were subjected to necropsy, which included weighing of major organs. Histopathology was conducted on tissues from five males from Control and High dose, and seven females from the Control and eight females from the High dose.

At 20000 ppm in-life observations included decreased weight gain and food consumption in both sexes. Increased male liver weight, 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 liver weight in males and alkaline phosphatase 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 20000 ppm with a corresponding decrease in the mean total number of pups born compared to 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. In conclusion, under the conditions of this study, toxicity was exhibited at levels of 5000 and 20000 ppm, but there were no clear effects of toxicity at 1000 ppm. Therefore the parental NOAEL was considered to be 1000 ppm. For reproductive parameters the NOEL was considered to be 5000 ppm.