Tall oil

Endpoint summary

Description of key information

Based on available data for groups of constituents present in Distilled Tall Oil, a conservative 2-year NOAEL of >200 mg/kg/d is set for the oral route.

An OECD 422 Combined Repeated Dose Toxicity Study with Reproductive/ Developmental Toxicity Screening Study is available for Distilled Tall Oil via the oral route in the diet for Sprague-Dawley rats (Inveresk Research, 2002e). The test was carried out according to the requirements of the guideline and in compliance with GLP. Effects on organ weights and clinical chemistry were observed at 5000 and 20,000 ppm in the diet, and decreased food consumption was also seen at the highest dose level, 20,000 ppm. A NOEL of 1000 ppm in the diet was established for general systemic toxicity in the study. Based on mean body weight and food consumption data over the duration of the study, this is equivalent to approximately 83 mg/kg/d for males and 100 mg/kg/d for females.

In a recently conducted OECD 408 repeated dose toxicity study in the rat (Charles River, 2020), administration of Tall Oil by dietary administration for at least 90 days was well tolerated in rats at levels up to 15,000 ppm and did not result in any adverse test item related (morphologic) alterations. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 15,000 ppm. This dietary level was equivalent to an overall mean daily intake of 1104 mg/kg/d in males and 1221 mg/kg/d in females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

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.

Qualifier:

according 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

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.

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

Route of administration:

oral: feed

Vehicle:

acetone

Details on oral 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).

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.

Dose / conc.:

0 ppm

Remarks:

Group 1 (control)

Dose / conc.:

1 000 ppm

Remarks:

Group 2

Dose / conc.:

5 000 ppm

Remarks:

Group 3

Dose / conc.:

20 000 ppm

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 positve control

Observations and examinations performed and frequency:

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:
- Animals fasted: No
- How many animals: Five males and five females
- Parameters checked in table [No.1] were examined.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2), all animals (males: after 4 weeks treatment; females: after Day 4 of lactation)
HISTOPATHOLOGY: Yes (see table 2), on control and high dose animals.

Other examinations:

No other examinations.

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.

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

Description (incidence and severity):

N/A

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Please see Table 14 & Table 15 for data.

In males, at 20000 ppm, there was a decrease in body weights, 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 20000 ppm attaining statistical significance.

In females, slight non-significant increases in liver weights following covariance analysis at 5000 and 20000 ppm were too small to attribute to treatment.

In males at 20000 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 20000 ppm, with pituitary gland weight reduced at 20000 ppm. Following covariance analysis, kidney and pituitary gland weights were essentially similar to Controls, but a decrease in ovary weight at 20000 ppm, and adrenal gland weight at 5000 and 20000 ppm, was still evident, but not significant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

N/A

Neuropathological findings:

not examined

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.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

N/A

Other effects:

not examined

Description (incidence and severity):

N/A

Details on results:

N/A

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 ppm

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.

Critical effects observed:

not specified

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

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.

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.

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 NOAEC was considered to be 1000 ppm.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25/03/19 - 24/09/19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Appendix to Director General Notification, No. 12-Nousan-8147

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Batch (Lot) Number: AN-400-125
Purity: 100%
Amber transparent liquid

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This strain is an accepted rodent species for preclinical toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were acclimatised for 13 days before the start of dosing. A clinical observation was undertaken during this period.

Animals were housed in a temperature range of 18-24 ºC, a relative target humidity of 40-70%, and a 12 hour light/dark cycle.

Rats were housed in groups up to 5 animals of the same sex and same dosing group together in polycarbonate cages.

Route of administration:

oral: feed

Details on route of administration:

The test and control items were administered by inclusion in the diet to the appropriate animals ad libitum from Day 1 for a minimum of 90 days, up to and including the day before scheduled necropsy. The first day diets were available to the animals was designated as Day 1.

The same diets remain in the food hopper for a maximum of one week. On the day of weighing the remaining food in the food hopper, remaining diet was replaced with new diet. The amount of test item incorporated into the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test article intake was estimated based on the body weight and food consumption values.

Vehicle:

acetone

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verification of doses was performed using a Agilent Technologies 7890B Gaschromatograph machine. The injection temperature was 260°C.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Test and control items were administered by inclusion in the diet to the appropriate animals ad libitum from day 1 for a minimum of 90 days- up to, and including the day before scheduled necropsy.

Dose / conc.:

0 ppm

Dose / conc.:

1 500 ppm

Dose / conc.:

5 000 ppm

Dose / conc.:

15 000 ppm

No. of animals per sex per dose:

10/sex/group

Control animals:

yes, concurrent vehicle

Positive control:

None

Observations and examinations performed and frequency:

Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical observations were performed once daily, once prior to first administration and beginning during the first administration of the test item and lasting up to the day prior to necropsy.

Animals were weighed individually weekly, starting on Day 1. A fasted weight was recorded on the day of necropsy

Food consumption was quantitatively measured weekly starting on Day 1 and continuing weekly throughout the Dosing Period.

Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol 5 mg/ml solution, THEA Pharma, Wetteren, Belgium) during Pretreatment in all Main Study animals, and at the end of the Dosing Period in Week 13 in all Group 1 and 4 Main study animals

Functional tests were performed on the first 5 animals per sex per group during Week 12-13. These tests were performed, after completion of clinical observations (including arena observation, if applicable).
Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
• Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

In the absence of a vaginal lavage, the study pathologist determined the estrous stage of all females by examining the reproductive organs.
These data were only used for evaluation of extreme thyroid hormone effects in females and are not included in the results section.

Sacrifice and pathology:

Blood was collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) for analysis. A blood smear was prepared from each hematology sample.

Haematology
Blood smears were labeled, stained, and stored. Blood smear from Animal No. 69 (5000 ppm) was evaluated to confirm analyser results.

Haematology parameters:
White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Reticulocyte (absolute), and Platelets

Coagulation
Blood samples at a target volume of 0.45 mL were collected into tubes containing citrate as anticoagulant. Samples were processed for plasma, and plasma was analyzed for prothrombin time and activated partial thromboplastin time

Clinical chemistry
Blood samples at a target volume of 0.5 mL were collected into tubes containing Li-heparinas anticoagulant. Samples were processed for plasma, and were analysed.
Blood samples at a target volume of 1.0 mL were collected in tubes without anticoagulant. Blood samples were processed for serum (bile acids and thyroid hormone measurement) and analysed

Clinical Chemistry parameters:
Alanine aminotransferase (ALAT) Glucose, Aspartate aminotransferase (ASAT), Cholesterol, Alkaline Phosphatase (ALP), HDL and LDL Cholesterol, Total protein, Triglyceridea (Triglyceride concentration determined from remaining thyroid hormone serum samples), Albumin, Sodium, Bile acids, Potassium, Total Bilirubin, Chloride, Urea, Calcium, Creatinine, and Inorganic Phosphate (Inorg. Phos)

Thyroid hormone
Serum samples at a target volume of 1.0 mL were collected in tubes without anticoagulant (same sample as for bile acid measurement). Blood samples were processed for serum, and were analysed.

Thyroid hormone parameters:
Triiodothyronine (T3), Thyroid-Stimulating Hormone (TSH), Thyroxine (T4)

Terminal procedures
Main study and recovery animals surviving until scheduled euthanasia were weighed, and euthanized using isoflurane, followed by exsanguination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy

Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

The following organ weights were weighed at necropsy for all scheduled euthanasia animals (paired organs were weighed together)
Brain, Epididymisa, Gland, adrenala, Gland, pituitary, Gland, prostate, Gland, seminal vesicle, Gland, thyroida, Heart, Kidney, Liver, Ovary, Spleen, Testis, Thymus, Uterus/Cervix
Organ to body weight ratio was calculated.

The following tissues were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands)

Artery, aorta, Body cavity, nasopharynx, Bone, femur, Bone marrow, Bone, sternum, Brain, Cervix, Epididymisa, Esophagus, Eye, adrenal gland, clitoral gland, harderian gland, lacrimal gland, mammary gland, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney, Large intestine, cecum, Large intestine, colon, Large intestine, rectum, Larynx, Liver, Lung, Lymph node, mandibular, Lymph node, mesenteric, skeletal muscle, optica nerve, sciatic nerve, Ovary, Pancreas, Skin, Small intestine, duodenum, Small intestine, ileum, Small intestine, jejunum, Spinal cord, Spleen, Stomach, Testisa, Thymus, Tongue, Trachea, Urinary bladder, Uterus, Vagina

All tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported. Tissues that were supposed to be microscopically evaluated per protocol but were not available on the slide (and therefore not evaluated) are listed in Individual Animal Data of the pathology report as not present. These missing tissues did/not affect the outcome or interpretation of the pathology portion of the study because sufficient data was available. A peer review on the histopathology data was performed by a second pathologist.

Statistics:

Body Weight Gains: Calculated against the body weight on Day 1.

Relative Food Consumption: Calculated between at least each scheduled interval.

Test Item Intake: Calculated as concentration of test item in diet against relative food consumption.

Organ Weight Relative to Body Weight: Calculated against the Terminal body weight

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), ratio, percentages, numbers, and/or incidences were reported as appropriate by dataset.

All statistical tests was conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations. The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-parametric
Datasets with at least 3 groups were compared using a Steel-test many-to-one rank test).

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Clinical signs, such as alopecia, scabs and incidental piloerection, noted during the Dosing Period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No effects on body weight and body weight gain were noted in males up to 5000 ppm and in females up to 15000 ppm.
A slightly lower body weight gain was observed in males at 15000 ppm throughout the administration period (-5% compared to controls).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level over the study period in males and females up to 15000 ppm.

Mean test article intake over the study period tabulated in "Any other information on results incl. tables" section.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no ophthalmology findings at Pretreatment and in Week 13. The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No effects on haematology parameters were noted in males at 1500 ppm and in females up to 15000 ppm.
Lower reticulocyte count was observed in males at 5000 ppm and 15000 ppm (0.83x and 0.84x of controls). In addition, red blood cell distribution width (RDW) was also minimally decreased in males at 15000 ppm (0.92x of controls). These changes werer just marginally outside historical control data, but in absence of any organ weight change or histopathological correlation these changes were not considered to be of toxicological significance.

While few other changes were statistically significant in males at 5000 and/or 15000 ppm, the alterations in haematological parameters were unrelated to administration of the test item due to the minimal magnitude of the change, remained within the historical control range, and/or absence of a clear dose response relationship.

Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No effects on clinical chemistry parameters were noted in males and females at 1500 and 5000 ppm.

At 15000 ppm, an increase in alkaline phosphatase (ALP) activity in males and females (1.53x and 2.06x of controls, respectively) and total bilirubin concentration in males (1.53x of controls) was observed. Furthermore, slight increases in total cholesterol and HDL and LDL cholesterol concentrations were observed in males at 15000 ppm (1.26x, 1.31x 1.60x of controls). In females at 15000 ppm, total cholesterol and HDL cholesterol were also slightly increased (1.16x and 1.28x of controls, respectively). Bile acids was also decreased in in males and females (0.54x and 0.41x of controls, respectively).

Total T3 was increased in males at 15000 ppm (1.50x of controls), which was caused by 4 males showing levels above the concurrent control range (maximally 2.41x of mean control value).

Other values in treated males and females achieving a levle of statistical significance when compared to controls where considered to have arisen as a result of slightly high or low control values, occured in the absence of a dose-related distribution and/or were, given the magnitude of change, considered to be of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength in animals up to 15000 ppm was similar to control, with the exception of the fore grip strength in males at 15000 ppm, which was slightly lower compared to control. However, as this fore grip strength remained well within the historical control range (633-1649 gram), this isolated finding was therefore considered not to be either test-material related or toxicologically relevant.

Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased absolute liver weight in females at 15000 (22%), and relative liver weight (to body weight) in males and females at 15000 (14 and 23% respectively)

There were no other test item-related organ weight changes. All other organ weight differences observed, were considered incidental and/or related to difference of sexual maturity and unrelated to the administration of Tall Oil.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.

All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with Tall Oil were noted at 15000 ppm in the liver of females and lung of both sexes.

Multifocal macrophage aggregation in the alveoli of the lung (minimal or slight) was recorded at increased incidence in males and females of the 15000 ppm group. The recorded incidences in the 1500 and/or 5000 ppm groups were comparable with the recorded incidences in the control group, or did not show a dose response and were therefore considered to be unrelated to treatment with the test item.

Hepatocellular hypertrophy of the liver was recorded at minimal degree in females of the 15000 ppm group.
Evaluation of the female reproductive organs for estrus stage revealed no abnormalities.
There were no other test item-related histologic changes. Remaining histologic changes (including the requested adrenal gland of males and kidneys of females) were considered to be within the range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects

Remarks on result:

other: approximate achieved doses of 1104 mg/kg/day in males and 1221 mg/kg/day in females

Key result

Critical effects observed:

no

Test Article Intake

Group	Nominal dietary inclusion level (ppm)	Mean over means intake (mg test item/kg body weight) (mean range indicated within brackets)
		Males	Females
2	1500	107 (79-153)	119 (94-152)
3	5000	367 (270-522)	420 (341-526)
4	15000	1104 (837-1506)	1221 (993-1520)

Summary of Body Weights (g) - Males

Treatment		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
Day 1 Week 1	Mean	177	176	173	179
	St. Dev.	11.7	10.0	7.9	5.7
	N	10	10	10	10
Day 8 Week 2	Mean	227	222	218	217
	St. Dev.	15.4	14.4	11.9	6.9
	N	10	10	10	10
Day 15 Week 3	Mean	265	261	260	257
	St. Dev.	18.6	20.0	15.9	8.0
	N	10	10	10	10
Day 22 Week 4	Mean	291	287	286	281
	St. Dev.	21.7	26.3	21.0	9.3
	N	10	10	10	10
Day 29 Week 5	Mean	315	311	315	308
	St. Dev.	23.4	31.5	23.8	10.0
	N	10	10	10	10
Day 36 Week 6	Mean	337	334	335	331
	St. Dev.	26.7	33.3	25.7	19.5
	N	10	10	10	10
Day 43 Week 7	Mean	354	350	352	346
	St. Dev.	29.2	35.4	28.7	10.9
	N	10	10	10	10
Day 50 Week 8	Mean	370	363	367	359
	St. Dev.	30.9	38.1	30.9	11.6
	N	10	10	10	10
Day 57 Week 9	Mean	378	371	376	365
	St. Dev.	31.1	37.9	34.2	12.2
	N	10	10	10	10
Day 64 Week 10	Mean	387	385	387	374
	St. Dev.	34.7	40.0	35.2	14.5
	N	10	10	10	10
Day 71 Week 11	Mean	397	391	393	383
	St. Dev.	34.3	40.9	36.4	15.7
	N	10	10	10	10
Day 78 Week 12	Mean	404	400	401	389
	St. Dev.	33.6	41.4	34.4	15.0
	N	10	10	10	10
Day 85 Week 13	Mean	415	407	411	399
	St. Dev.	34.7	38.9	35.2	14.1
	N	10	10	10	10
Day 91 Week 13	Mean	416	408	406	397
	St. Dev.	35.1	40.0	35.7	16.5
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Summary of Body Weights (g) - Females

Treatment		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
Day 1 Week 1	Mean	131	132	137	132
	St. Dev.	4.9	8.2	7.1	7.4
	N	10	10	10	10
Day 8 Week 2	Mean	150	153	158	149
	St. Dev.	8.4	8.8	7.1	10.5
	N	10	10	10	10
Day 15 Week 3	Mean	169	173	178	166
	St. Dev.	8.2	8.6	7.8	10.4
	N	10	10	10	10
Day 22 Week 4	Mean	180	183	191*	177
	St. Dev.	9.3	8.9	7.1	10.1
	N	10	10	10	10
Day 29 Week 5	Mean	193	194	202	190
	St. Dev.	9.7	10.1	7.4	9.0
	N	10	10	10	10
Day 36 Week 6	Mean	200	205	211	200
	St. Dev.	12.3	10.2	10.7	8.5
	N	10	10	10	10
Day 43 Week 7	Mean	212	215	220	206
	St. Dev.	10.8	11.6	10.3	11.8
	N	10	10	10	10
Day 50 Week 8	Mean	218	223	224	212
	St. Dev.	12.5	11.8	10.4	13.0
	N	10	10	10	10
Day 57 Week 9	Mean	219	222	226	213
	St. Dev.	11.9	14.1	9.2	9.5
	N	10	10	10	10
Day 64 Week 10	Mean	219	226	231	216
	St. Dev.	12.7	12.6	12.1	9.7
	N	10	10	10	10
Day 71 Week 11	Mean	223	232	234	220
	St. Dev.	11.6	13.1	11.6	11.4
	N	10	10	10	10
Day 78 Week 12	Mean	229	234	238	223
	St. Dev.	12.3	13.4	10.9	10.7
	N	10	10	10	10
Day 85 Week 13	Mean	232	234	242	227
	St. Dev.	12.5	14.8	11.6	12.9
	N	10	10	10	10
Day 91 Week 13	Mean	227	235	240	225
	St. Dev.	11.3	14.2	11.4	10.8
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Summary of Body Weight Gain (%) - Males

Treatment		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
Day 1 Week 1	Mean	0	0	0	0
	St. Dev.	0.0	0.0	0.0	0.0
	N	10	10	10	10
Day 8 Week 2	Mean	28	26	26	21**
	St. Dev.	1.9	2.7	1.7	3.0
	N	10	10	10	10
Day 15 Week 3	Mean	50	48	50	43**
	St. Dev.	3.7	4.9	3.6	4.6
	N	10	10	10	10
Day 22 Week 4	Mean	64	62	65	57*
	St. Dev.	3.9	8.9	5.8	5.8
	N	10	10	10	10
Day 29 Week 5	Mean	78	76	82	72
	St. Dev.	5.6	12.6	7.7	7.1
	N	10	10	10	10
Day 36 Week 6	Mean	90	89	93	85
	St. Dev.	6.6	13.1	8.5	10.2
	N	10	10	10	10
Day 43 Week 7	Mean	100	98	103	93
	St. Dev.	7.0	14.4	10.5	7.9
	N	10	10	10	10
Day 50 Week 8	Mean	109	106	111	101
	St. Dev.	8.4	15.8	11.8	8.5
	N	10	10	10	10
Day 57 Week 9	Mean	113	110	117	104
	St. Dev.	9.0	16.1	13.1	9.5
	N	10	10	10	10
Day 64 Week 10	Mean	118	118	123	109
	St. Dev.	10.3	17.3	13.8	10.4
	N	10	10	10	10
Day 71 Week 11	Mean	124	122	126	114
	St. Dev.	10.6	17.6	14.4	10.7
	N	10	10	10	10
Day 78 Week 12	Mean	128	127	131	117
	St. Dev.	10.5	17.5	14.2	10.4
	N	10	10	10	10
Day 85 Week 13	Mean	134	131	137	123
	St. Dev.	11.5	16.4	14.3	10.3
	N	10	10	10	10
Day 91 Week 13	Mean	135	131	134	121
	St. Dev.	10.8	16.5	14.6	10.9
	N	10	10	10	10

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Summary of Body Weight Gain (%) - Females

Treatment		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
Day 1 Week 1	Mean	0	0	0	0
	St. Dev.	0.0	0.0	0.0	0.0
	N	10	10	10	10
Day 8 Week 2	Mean	14	17	16	12
	St. Dev.	3.5	3.4	3.3	3.3
	N	10	10	10	10
Day 15 Week 3	Mean	29	32	30	26
	St. Dev.	3.1	3.3	4.4	3.2
	N	10	10	10	10
Day 22 Week 4	Mean	37	39	39	34
	St. Dev.	3.4	4.9	5.1	3.1
	N	10	10	10	10
Day 29 Week 5	Mean	47	47	47	43
	St. Dev.	3.7	5.2	4.6	5.5
	N	10	10	10	10
Day 36 Week 6	Mean	52	56	54	51
	St. Dev.	5.2	6.9	7.4	4.8
	N	10	10	10	10
Day 43 Week 7	Mean	62	64	61	56
	St. Dev.	4.4	7.4	7.5	8.0
	N	10	10	10	10
Day 50 Week 8	Mean	66	70	63	60
	St. Dev.	5.9	8.8	6.6	8.6
	N	10	10	10	10
Day 57 Week 9	Mean	67	69	65	61
	St. Dev.	4.9	9.8	6.2	6.5
	N	10	10	10	10
Day 64 Week 10	Mean	66	72	69	63
	St. Dev.	6.6	7.7	8.1	6.1
	N	10	10	10	10
Day 71 Week 11	Mean	70	77	71	67
	St. Dev.	5.3	8.6	8.5	6.5
	N	10	10	10	10
Day 78 Week 12	Mean	74	78	74	69
	St. Dev.	5.8	7.8	7.3	8.1
	N	10	10	10	10
Day 85 Week 13	Mean	77	78	77	72
	St. Dev.	6.9	7.6	7.5	9.0
	N	10	10	10	10
Day 91 Week 13	Mean	73	78	76	70
	St. Dev.	5.7	7.8	9.5	7.2
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Summary of Ophthalmoscopic Observations

	Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
Males
Pre-treatment
No findings	8/10	10/10	10/10	10/10
Persistent Pupillary Membrane	2/10	0/10	0/10	0/10
End of Treatment
No findings	10/10			10/10
Females
Pre-treatment
No findings	10/10	10/10	9/10	9/10
Focal Corneal Opacity	0/10	0/10	1/10	1/10
End of Treatment
No findings	10/10			10/10

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Haematology summary – Males

End of treatment		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
WBC 10E9/L	Mean	7.2	6.2	6.3	5.8
	St. Dev	1.4	1.6	1.7	1.6
	N	10	10	10	9
Neutrophils 10E8/L	Mean	1.0	0.9	0.8	0.8
	St. Dev	0.4	0.3	0.3	0.4
	N	10	10	10	9
Lymphocytes 10E9/L	Mean	5.9	5.1	5.3	4.8
	St. Dev	1.2	1.5	1.5	1.3
	N	10	10	10	9
Monocytes 10E9/L	Mean	0.1	0.1	0.1	0.1
	St. Dev	0.1	0.0	0.1	0.0
	N	10	10	10	9
Eosinophils 10E9/L	Mean	0.1	0.1	0.1	0.1
	St. Dev	0.0	0.0	0.0	0.1
	N	10	10	10	9
Basophils 10E9/L	Mean	0.0	0.0	0.0	0.0
	St. Dev	0.0	0.0	0.0	0.0
	N	10	10	10	9
Red Blood Cells 10E12/L	Mean	9.08	8.94	8.90	8.85
	St. Dev	0.42	0.24	0.42	0.33
	N	10	10	10	9
Reticulocytes 10E9/L	Mean	165.2	164.4	137.0	139.8
	St. Dev	30.5	24.2	45.0	20.1
	N	10	10	10	9
RDW %	Mean	11.9	11.6	11.4	11.0**
	St. Dev	0.6	0.4	0.8	0.3
	N	10	10	10	9
Haemoglobin Mmol/L	Mean	10.3	10.1	9.9*	9.7**
	St. Dev	0.4	0.2	0.3	0.2
	N	10	10	10	9
Haematocrit L/L	Mean	0.477	0.470	0.464	0.460
	St. Dev	0.017	0.010	0.018	0.012
	N	10	10	10	9
MCV fL	Mean	52.5	52.6	52.1	52.0
	St. Dev	1.5	0.9	0.8	1.1
	N	10	10	10	9
MCH Fmol	Mean	1.13	1.13	1.12	1.10
	St. Dev	0.05	0.03	0.03	0.03
	N	10	10	10	9
MCHC Mmol/L	Mean	21.54	21.47	21.39	21.05*
	St. Dev	0.52	0.42	0.37	0.30
	N	10	10	10	9
Platelets 10E9/L	Mean	710	680	680	723
	St. Dev	70	75	75	81
	N	10	10	10	9
PT s	Mean	17.8	17.1	17.6	17.8
	St. Dev	0.9	0.6	1.0	1.0
	N	10	10	10	9
APTT s	Mean	17.7	17.9	18.1	16.7
	St. Dev	3.4	2.8	2.7	3.5
	N	10	10	10	9

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Haematology summary – Females

End of treatment		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
WBC 10E9/L	Mean	6.7	4.3**	3.9**	5.4
	St. Dev	1.6	1.4	1.3	1.1
	N	10	10	10	10
Neutrophils 10E8/L	Mean	0.7	0.6	0.5	0.6
	St. Dev	0.2	0.2	0.2	0.3
	N	105.8	10	10	10
Lymphocytes 10E9/L	Mean	1.5	3.6 +	3.3++	4.6
	St. Dev	0.1	1.2	1.1	1.1
	N	10	10	10	10
Monocytes 10E9/L	Mean	0.1	0.1 +	0.1	0.1
	St. Dev	0.0	0.0	0.0	0.0
	N	10	10	10	10
Eosinophils 10E9/L	Mean	0.1	0.1	0.1	0.1
	St. Dev	0.0	0.0	0.0	0.0
	N	10	10	10	10
Basophils 10E9/L	Mean	0.0	0.0	0.0	0.0
	St. Dev	0.0	0.0	0.0	0.0
	N	10	10	10	10
Red Blood Cells 10E12/L	Mean	8.28	7.91*	8.11	8.14
	St. Dev	0.26	0.21	0.36	0.36
	N	10	10	10	10
Reticulocytes 10E9/L	Mean	187.8	195.0	165.2	158.3
	St. Dev	29.4	25.8	15.7	23.3
	N	10	10	10	10
RDW %	Mean	10.3	10.4	10.3	10.2
	St. Dev	0.3	0.2	0.4	0.5
	N	10	10	10	10
Haemoglobin Mmol/L	Mean	9.5	9.0 **	9.2*	9.2
	St. Dev	0.3	0.3	0.3	0.3
	N	10	10	10	10
Haematocrit L/L	Mean	0.118	0.433*	0.434	0.435
	St. Dev	0.014	0.012	0.011	0.015
	N	10	10	10	10
MCV fL	Mean	54.1	54.7	53.6	53.6
	St. Dev	0.9	1.2	1.9	1.3
	N	10	10	10	10
MCH Fmol	Mean	1.15	1.14	1.14	1.14
	St. Dev	0.03	0.03	0.06	0.03
	N	10	10	10	10
MCHC Mmol/L	Mean	21.26	20.84	21.24	21.21
	St. Dev	0.38	0.39	0.40	0.35
	N	10	10	10	10
Platelets 10E9/L	Mean	835	7.3*	707*	795
	St. Dev	139	69	114	105
	N	10	10	10	10
PT s	Mean	17.7	17.4	17.2	17.3
	St. Dev	0.3	0.8	0.7	0.6
	N	10	10	10	10
APTT s	Mean	18.5	16.9	17.0	17.0
	St. Dev	1.2	2.3	2.1	2.0
	N	10	10	10	10

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Clinical Biochemistry Summary – Males

End of treatment		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
ALAT U/L	Mean	46.2	47.0	48.9	54.0
	St. Dev	8.4	10.8	8.8	15.0
	N	10	10	10	10
ASAT U/L	Mean	84.1	79.9	81.2	82.6
	St. Dev	13.8	10.6	936	7.7
	N	10	10	10	10
ALP U/L	Mean	130	125	155	199**
	St. Dev	45	24	43	42
	N	10	10	10	10
Total Protein g/L	Mean	66.6	65.6	64.4	65.6
	St. Dev	2.0	1.8	2.1	2.0
	N	10	10	10	10
Albumin g/L	Mean	33.5	33.5	32.7	33.3
	St. Dev	0.9	1.1	1.1	0.6
	N	10	10	10	10
Total Bilirubin μmol/L	Mean	2.9	2.9	3.3	3.5*
	St. Dev	0.4	0.4	0.5	0.6
	N	10	10	10	10
Urea mmol/L	Mean	7.4	7.9	7.2	8.0
	St. Dev	1.4	1.7	1.9	0.9
	N	10	10	10	10
Creatinine μmol/L	Mean	39.7	42.7**	41.9	44.4**
	St. Dev	1.9	1.7	2.5	1.8
	N	10	10	10	10
Glucose mmol/L	Mean	11.89	11.02	10.07*	10.46
	St. Dev	2.00	1.66	1.29	1.26
	N	10	10	10	10
Cholesterol mmol/L	Mean	2.29	2.31	2.29	2.88**
	St. Dev	0.28	0.23	0.21	0.43
	N	10	10	10	10
HDL cholesterol mmol/L	Mean	0.94	0.97	0.89	1.23**
	St. Dev	0.12	0.14	0.10	0.15
	N	10	10	10	10
LDL Cholesterol mmol/L	Mean	0.30	0.30	0.30	0.48**
	St. Dev	0.04	0.04	0.05	0.11
	N	10	10	10	10
Triglycerides mmol/L	Mean	1.16	0.86	1.00	0.78*
	St. Dev	0.21	0.15	0.43	0.23
	N	10	10	10	10
Bile Acids μmole/L	Mean	20.3	25.3	15.5	10.9*
	St. Dev	6.1	10.8	5.6	4.5
	N	10	10	10	10
Sodium mmol/L	Mean	140.6	141.7*	141.8*	140.5
	St. Dev	0.9	0.6	1.1	1.0
	N	10	10	10	10
Potassium mmol/L	Mean	3.84	3.85	3.97	4.01
	St. Dev	0.12	0.0	0.19	0.22
	N	10	10	10	10
Chloride mmol/L	Mean	102	103*	103**	103*
	St. Dev	0	1	1	1
	N	10	10	10	10
Calcium mmol/L	Mean	2.66	2.58*	2.61	2.64
	St. Dev	0.07	0.04	0.04	0.08
	N	10	10	10	10
Inorg. Phos mmol/L	Mean	1.56	1.47	1.53	1.57
	St. Dev	0.20	0.14	0.12	0.21
	N	10	10	10	10
TSH ulU/mL	Mean	0.129	0.309*	0.201	0.16.
	St. Dev	0.134	0.192	0.198	0.094
	N	10	10	10	10
Total T3 ng/dL	Mean	47.4	47.6	53.4	71.1*
	St. Dev	4.4	4.4	10.2	25.3
	N	10	10	10	10
Total T4 μg/dL	Mean	4.73	4.51	4.30	4.70
	St. Dev	0.59	0.77	0.73	1.12
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Clinical Biochemistry Summary – Females

End of treatment		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
ALAT U/L	Mean	48.7	36.5	43.2	52.2
	St. Dev	14.0	7.9	13.6	15.4
	N	10	10	10	10
ASAT U/L	Mean	81.5	77.5	87.4	81.0
	St. Dev	5.8	4.4	7.1	8.1
	N	10	10	10	10
ALP U/L	Mean	53	49	59	109**
	St. Dev	14	12	15	54
	N	10	10	10	10
Total Protein g/L	Mean	69.4	69.1	68.1	68.1
	St. Dev	1.9	2.2	2.2	2.2
	N	10	10	10	10
Albumin g/L	Mean	36.8	36.4	36.3	35.8
	St. Dev	1.3	1.8	1.3	1.4
	N	10	10	10	10
Total Bilirubin μmol/L	Mean	3.3	3.5	3.6	3.7
	St. Dev	0.4	0.4	0.7	0.3
	N	10	10	10	10
Urea mmol/L	Mean	7.3	7.4	7.2	7.6
	St. Dev	1.4	1.1	0.9	1.0
	N	10	10	10	10
Creatinine μmol/L	Mean	38.8	45.4**	43.2*	43.3*
	St. Dev	3.0	4.1	3.8	2.7
	N	10	10	10	10
Glucose mmol/L	Mean	9.27	8.87	8.20	8.63
	St. Dev	1.79	1.53	0.90	1.26
	N	10	10	10	10
Cholesterol mmol/L	Mean	2.17	2.20	2.22	2.51
	St. Dev	0.30	0.35	0.37	0.22
	N	10	10	10	10
HDL cholesterol mmol/L	Mean	0.94	0.95	0.98	1.20**
	St. Dev	0.17	0.19	0.21	0.14
	N	10	10	10	10
LDL Cholesterol mmol/L	Mean	0.28	0.28	0.26	0.32
	St. Dev	0.04	0.06	0.05	0.02
	N	10	10	10	10
Triglycerides mmol/L	Mean	0.66	0.59	0.74	0.79
	St. Dev	0.27	0.16	0.25	0.21
	N	10	10	10	10
Bile Acids μmole/L	Mean	19.2	13.2	15.2	7.9**
	St. Dev	13.1	5.4	4.1	3.6
	N	10	10	10	10
Sodium mmol/L	Mean	140.2	140.7	140.6	140.4
	St. Dev	0.7	0.6	1.0	0.4
	N	10	10	10	10
Potassium mmol/L	Mean	3.77	3.45*	3.53*	3.57
	St. Dev	0.14	0.22	0.17	0.16
	N	10	10	10	10
Chloride mmol/L	Mean	103	105**	104*	104
	St. Dev	1	1	1	1
	N	10	10	10	10
Calcium mmol/L	Mean	2.72	2.60**	2.59**	2.71
	St. Dev	0.04	0.06	0.04	0.03
	N	10	10	10	10
Inorg. Phos mmol/L	Mean	1.56	1.14**	1.11**	1.51
	St. Dev	0.13	0.19	0.14	0.19
	N	10	10	10	10
TSH ulU/mL	Mean	0.036	0.119*	0.103	0.109*
	St. Dev	0.015	0.089	0.064	0.073
	N	10	10	10	10
Total T3 ng/dL	Mean	49.2	51.6	49.1	43.7
	St. Dev	7.9	7.8	4.8	2.3
	N	10	10	10	10
Total T4 μg/dL	Mean	1.95	2.94*	2.55	2.37
	St. Dev	0.63	1.01	0.77	0.049
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Summary of Macroscopic Findings

	Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
Males
End of Treatment
Animals examined	10	10	10	10
Animals without findings	6	9	8	6
Animals affected	4	1	2	4
Stomach: Thickened	1	0	0	0
Seminal vesicles: Reduced size	2	1	1	0
Preputial glands: Focus/Foci	0	0	0	1
Thyroid gland: Discolouration	0	0	0	1
Thymus: Focus/Foci	1	0	1	1
Mandibular lymph Node: Enlarged	0	0	0	1
Females
End of Treatment
Animals examined	10	10	10	10
Animals without findings	8	6	6	7
Animals affected	2	4	4	3
Kidneys: Pelvic dilation	0	0	1	0
Uterus: Contains fluid	1	2	4	2
Clitoral glands: Focus/Foci	0	1	0	1
Thymus: Focus/Foci	0	1	0	0
Mandibular lymph Node: Discolouration	0	0	0	1
Eyes: Exophthalmus	0	1	0	0
Body cavities: Nodule(s)	1	0	0	0

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level    

Summary of Organ Weights (g) - Males

		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
End of Treatment
Body Weight (g)	Mean	396	387	384	382
	St. Dev.	34	38	33	18
	N	10	10	10	10
Brain (g)	Mean	2.04	2.07	2.06	2.07
	St. Dev.	0.07	0.08	0.07	0.07
	N	10	10	10	10
Pituitary (g)	Mean	0.009	0.009	0.009	0.009
	St. Dev.	0.001	0.001	0.002	0.001
	N	10	10	10	10
Heart (g)	Mean	1.020	1.018	0.968	0.976
	St. Dev.	0.086	0.110	0.075	0.036
	N	10	10	10	10
Liver (g)	Mean	9.32	9.19	8.91	10.18
	St. Dev.	1.24	1.42	1.09	0.86
	N	10	10	10	10
Thyroids (g)	Mean	0.018	0.019	0.021	0.020
	St. Dev.	0.002	0.005	0.005	0.003
	N	10	10	10	10
Thymus (g)	Mean	0.301	0.258	0.264	0.300
	St. Dev.	0.070	0.083	0.063	0.065
	N	10	10	10	10
Kidneys (g)	Mean	2.38	2.39	2.39	2.40
	St. Dev.	0.24	0.28	0.19	0.19
	N	10	10	10	10
Adrenals (g)	Mean	0.056	0.053	0.055	0.051
	St. Dev.	0.006	0.007	0.004	0.006
	N	10	10	10	10
Spleen (g)	Mean	0.569	0.577	0.580	0.553
	St. Dev.	0.059	0.085	0.063	0.053
	N	10	10	10	10
Testes (g)	Mean	3.68	3.66	3.62	3.64
	St. Dev.	0.30	0.30	0.25	0.17
	N	10	10	10	10
Prostate (g)	Mean	0.908	0.805	0.843	0.790
	St. Dev.	0.149	0.188	0.101	0.135
	N	10	10	10	10
Epididymides (g)	Mean	1.195	1.187	1.147	1.178
	St. Dev.	0.085	0.084	0.091	0.053
	N	10	10	10	10
Seminal Vesicle (g)	Mean	1.219	1.251	1.223	1.300
	St. Dev.	0.181	0.220	0.212	0.616
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Summary of Organ / Body weight Ratios (%) - Males

		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
End of Treatment
Body Weight (g)	Mean	396	387	384	382
	St. Dev.	34	38	33	18
	N	10	10	10	10
Brain (%)	Mean	0.52	0.54	0.54	0.54
	St. Dev.	0.04	0.04	0.04	0.03
	N	10	10	10	10
Pituitary (%)	Mean	0.002	0.002	0.002	0.002
	St. Dev.	0.000	0.000	0.000	0.000
	N	10	10	10	10
Heart (%)	Mean	0.258	0.263	0.253	0.255
	St. Dev.	0.012	0.010	0.012	0.008
	N	10	10	10	10
Liver (%)	Mean	2.34	2.37	2.32	2.66**
	St. Dev.	0.17	0.18	0.14	0.18
	N	10	10	10	10
Thyroids (%)	Mean	0.005	0.005	0.005	0.005
	St. Dev.	0.000	0.001	0.001	0.001
	N	10	10	10	10
Thymus (%)	Mean	0.076	0.067	0.069	0.079
	St. Dev.	0.016	0.018	0.016	0.017
	N	10	10	10	10
Kidneys (%)	Mean	0.60	0.62	0.62	0.63
	St. Dev.	0.02	0.03	0.03	0.04
	N	10	10	10	10
Adrenals (%)	Mean	0.014	0.014	0.014	0.013
	St. Dev.	0.002	0.002	0.001	0.002
	N	10	10	10	10
Spleen (%)	Mean	0.144	0.149	0.151	0.145
	St. Dev.	0.018	0.019	0.015	0.012
	N	10	10	10	10
Testes (%)	Mean	0.93	0.96	0.95	0.96
	St. Dev.	0.10	0.13	0.09	0.06
	N	10	10	10	10
Prostate (%)	Mean	0.230	0.208	0.220	0.207
	St. Dev.	0.041	0.043	0.025	0.038
	N	10	10	10	10
Epididymides	Mean	0.304	0.309	0.300	0.309
	St. Dev.	0.038	0.036	0.024	0.022
	N	10	10	10	10
Seminal Vesicle (%)	Mean	0.309	0.325	0.320	0.341
	St. Dev.	0.054	0.058	0.059	0.047
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Summary of Organ Weights (g) - Females

		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
End of Treatment
Body Weight (g)	Mean	217	223	229	214
	St. Dev.	11	14	10	9
	N	10	10	9	10
Brain (g)	Mean	1.89	1.88	1.89	1.89
	St. Dev.	0.08	0.05	0.06	0.08
	N	10	10	10	10
Pituitary (g)	Mean	0.013	0.014	0.012	0.011
	St. Dev.	0.002	0.004	0.002	0.003
	N	10	10	10	10
Heart (g)	Mean	0.704	0.729	0.710	0.654
	St. Dev.	0.066	0.087	0.051	0.042
	N	10	10	10	10
Liver (g)	Mean	5.27	5.66	5.70	6.42**
	St. Dev.	0.40	0.58	0.40	0.53
	N	9	10	10	10
Thyroids (g)	Mean	0.014	0.015	0.015	0.014
	St. Dev.	0.003	0.002	0.003	0.022
	N	10	10	10	10
Thymus (g)	Mean	0.270	0.264	0.275	0.269
	St. Dev.	0.035	0.036	0.055	0.055
	N	10	10	10	10
Kidneys (g)	Mean	1.53	1.52	1.55	1.50
	St. Dev.	0.12	0.11	0.11	0.12
	N	10	10	10	10
Adrenals (g)	Mean	0.067	0.070	0.065	0.058
	St. Dev.	0.008	0.007	0.011	0.008
	N	10	10	10	10
Spleen (g)	Mean	0.377	0.422	0.390	0.393
	St. Dev.	0.039	0.051	0.047	0.043
	N	10	10	10	10
Ovaries (g)	Mean	0.133	0.145	0.148	0.140
	St. Dev.	0.020	0.021	0.018	0.021
	N	10	10	10	10
Uterus (g)	Mean	0.669	0.628	0.765	0.662
	St. Dev.	0.400	0.199	0.389	0.445
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Summary of Organ / Body weight Ratios (%) - Females

		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
End of Treatment
Body Weight (g)	Mean	217	223	229	214
	St. Dev.	11	14	10	9
	N	10	10	9	10
Brain (%)	Mean	0.87	0.85	0.83	0.88
	St. Dev.	0.04	0.05	0.03	0.03
	N	10	10	9	10
Pituitary (%)	Mean	0.006	0.006	0.005	0.005
	St. Dev.	0.001	0.002	0.001	0.001
	N	10	10	9	10
Heart (%)	Mean	0.326	0.327	0.314	0.305
	St. Dev.	0.037	0.042	0.013	0.015
	N	10	10	9	10
Liver (%)	Mean	2.44	2.54	2.52	3.00**
	St. Dev.	0.17	0.20	0.12	0.28
	N	9	10	9	10
Thyroids (%)	Mean	0.006	0.007	0.007	0.007
	St. Dev.	0.001	0.001	0.001	0.001
	N	10	10	9	10
Thymus (%)	Mean	0.124	0.118	0.125	0.126
	St. Dev.	0.015	0.014	0.020	0.027
	N	10	10	9	10
Kidneys (%)	Mean	0.70	0.68	0.69	0.70
	St. Dev.	0.06	0.05	0.04	0.06
	N	10	10	9	10
Adrenals (%)	Mean	0.031	0.031	0.028	0.027
	St. Dev.	0.003	0.003	0.004	0.004
	N	10	10	9	10
Spleen (%)	Mean	0.174	0.189	0.169	0.183
	St. Dev.	0.016	0.022	0.019	0.107
	N	10	10	9	10
Ovaries (%)	Mean	0.061	0.065	0.064	0.065
	St. Dev.	0.010	0.011	0.006	0.008
	N	10	10	9	10
Uterus (%)	Mean	0.307	0.283	0.344	0.309
	St. Dev.	0.177	0.093	0.167	0.206
	N	10	10	9	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Mean percentage liver weight differences from control groups

Dose level (ppm)	Males			Females
	1500	5000	15000	1500	5000	15000
LIVER
Absolute	-1	-4	9	7	8	22**
Relative to body weight	1	-1	14**	4	3	23**

*: P<0.05, **:P<0.01

Summary of Treatment-Related Microscopic Finding

	Males				Females
Dose Level (ppm)	0	1500	5000	15000	0	1500	5000	15000
Lung^a	10	10	10	10	10	10	10	10
Macrophage aggregation, multifocal
Minimal	1	-	1	3	2	1	2	6
Slight	-	-	1	3	-	-	1	-
Liver^a	10	10	10	10	10	10	10	10
Hepatocellular hypertrophy
Minimal	-	-	-	-	-	-	-	4

^a Number of tissues examined from each group.

Summary of Functional Observations

		Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
Males
End of Treatment
Hearing Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Pupil L Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Pupil R Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Static R Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Grip Fore Gram	Mean	1263	1211	1094	956*
	St. Dev	61	270	218	120
	N	5	5	5	5
Grip Hind Gram	Mean	493	592	515	520
	St. Dev	62	115	47	76
	N	5	5	5	5
Females
End of Treatment
Hearing Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Pupil L Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Pupil R Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Static R Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Grip Fore Gram	Mean	980	850	747	882
	St. Dev	129	102	147	182
	N	5	5	5	5
Grip Hind Gram	Mean	363	381	396	422
	St. Dev	41	68	93	60
	N	5	5	5	5

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Summary of Motor Activity

	Group 1 Control	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15000 ppm
Males
Total Movements
Mean	4535	4340	3552	4174
N	5	5	5	5
St. Dev	929	1039	731	372
Ambulations
Mean	950	924	697	846
N	5	5	5	5
St. Dev	385	264	272	112
Females
Total Movements
Mean	4536	5543	4666	4490
N	5	5	5	5
St. Dev	866	766	927	1538
Ambulations
Mean	1224	1530	1140	1208
N	5	5	5	5
St. Dev	294	315	321	612

* indicates a p-value <0.05, ** indicates a p-value <0.01

MEAN and ST. DEV values are calculated per group, from each animal's Total Movements over all interval

 

Conclusions:

In conclusion, administration of Tall Oil by dietary administration for at least 90 days was generally well tolerated in Han Wistar rats at levels up to 15000 ppm and did not result in any adverse test item-related (morphologic) alterations.

Therefore, based on these results, the No-Observed-Adverse-Effect Level (NOAEL) was considered to be 15000 ppm (which equates to approximate achieved doses of 1104 mg/kg/day in males and 1221 mg/kg/day in females).

Executive summary:

The objective of this study was to determine the potential toxicity of Tall Oil, when given via diet administration for 90 days to Wistar Han rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.

 

The study design was as follows:

 

Table 1: Experimental Design

Group No.	Test Item ID.	Dose level (ppm)	No. of Animals
			Male		Female
1	-	0 (Vehicle)	10	10
2	Tall Oil	1500	10	10
3		5000	10	10
4		15000	10	10

Table 2: Test Article Intake

Group	Nominal dietary inclusion level (ppm)	Mean over means intake (mg test item/kg body weight) (mean range indicated within brackets)
		Males	Females
2	1500	107 (79-153)	119 (94-152)
3	5000	367 (270-522)	420 (341-526)
4	15000	1104 (837-1506)	1221 (993-1520)

 

Chemical analyses of dietary preparations were conducted three times to assess accuracy and homogeneity. Additional analysis for stability was done in Week 13.

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, ophthalmology, functional observations, estrous stage determination, clinical pathology parameters (hematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations. Dietary analyses confirmed that formulations of test item in diets were prepared accurately and homogenously in Weeks 1 and 6. The concentrations analyzed in the diets prepared in Week 13 were not in agreement with target concentrations (i.e. mean accuracies between 73 and 79%). Therefore, as concentrations in Week 13 were only below the acceptable range (80%-120%) for up to a maximum of 7% and taken into account that the Weeks 1 and 6 diets which were analyzed were within specification, it was concluded that the diets were accurately prepared throughout the administration period.

At 1500 ppm, no test item-related changes were observed.

At 5000 ppm, the only observed test item-related finding was lower reticulocyte count in males which was, at the severity observed and the absence of histopathological correlation, considered not to be adverse.

At 15000 ppm, multiple test item-related effects were seen, which were considered not to be adverse. Firstly, a lower body weight gain was observed in males throughout the administration period. Affected test item-related hematology changes consisted of slightly lower reticulocyte count and decreased red blood cell distribution width in males. The body weight and hematology effects were only slight and in absence of a histopathological correlation considered not to be adverse. 

At clinical chemistry, an increase in alkaline phosphatase activity was noted in males and females, which in females correlates with the hepatocellular hypertrophy of the liver. Furthermore, increases in total bilirubin concentration in males and total cholesterol and HDL and/or LDL cholesterol concentrations in males and females and decreases in bile acids concentration in males and females were observed. Finally, total T3 was increased in males at 15000 ppm. The bilirubin, cholesterol, bile acids and T3 findings were in absence of a histopathological correlation or any effects in the other thyroid hormones considered not to be adverse. At histopathological examination, minimal hepatocellular hypertrophy of the liver was observed in females, which correlated with increased liver weights and higher alkaline phosphatase activity and total bilirubin concentration. There was no microscopic correlate for the observed increased liver weight in males. These findings were at the observed severity and/or magnitude and in absence of any concomitant degenerative finding considered nonadverse and is a commonly observed adaptive response associated with the metabolism of xenobiotics or their metabolites. In the lung, multifocal macrophage aggregation was observed in males and females. The exacerbation in incidence of this finding at low degree of both sexes was regarded as test item-related and in absence of any other test item-related lung finding, was considered not to be adverse.

No test item-related or toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, food consumption, functional observations, ophthalmoscopy, coagulation parameters and macroscopic examination). In conclusion, administration of Tall Oil by dietary administration for at least 90 days was generally well tolerated in Han Wistar rats at levels up to 15000 ppm and did not result in any adverse test item-related (morphologic) alterations. Therefore, based on these results, the No-Observed-Adverse-Effect Level (NOAEL) was considered to be 15000 ppm (which equates to approximate achieved doses of 1104 mg/kg/day in males and 1221 mg/kg/day in females).

Endpoint conclusion

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In addition, data are also available for the constituent parts of DTO and related substances (unpublished laboratory studies and information published in the public domain). These are as follows -

Fatty acids: Three repeated dose toxicity studies are reported under the US EPA HPV program for TOFA. The study of longest duration, 90-days, is considered to be the key study for this endpoint. During the 90 day study administration of up to 25% (approximately 12,500 mg/kg bw/day) Tall Oil Fatty Acids (TOFA) in the diet of Charles River rats daily for 90 days did not produce any adverse effects. The NOAEL is greater than or equal to 12,500 mg/kg bw/day.

Resin acids and neutrals: A number of repeated dose toxicity studies (90-day and 2-year) are available in rats and dogs for Tall Oil Rosin and other related types of rosin. Although these tests do not meet current test guidelines in all respects, the overall lack of toxicity in two species with all product types indicates that further testing would not be appropriate. Only the rat data are discussed further here, since this is the preferred species for testing according to REACH. The following oral dietary studies have been performed: Tall oil Rosin (90 day, Calandra, 1960a, Kay 1962a), Gum rosin (90 day & 2 year, calandra 1960c, Kay 1962b), n-wood rosin (90 day & 2 year study, Calandra 1960d, Kay 1962c).Studies were performed at dose levels up to 1.0% in the diet, prepared as a suspension in corn oil prior to mixing with feed.

Examinations performed were: Body weight gain, mortality and abnormal behavioural reactions on a daily basis; Haematological studies and urine analysis; Liver and kidney function tests, gross and microscopic pathologic studies (comprising heart, aorta, trachea, lungs, liver, gall bladder, pancreas, oesophagus, stomach, small & large intestine, spleen, lymph nodes, kidney, urinary bladder, gonads, prostate, uterus, pituitary, adrenal gland, salivary gland, thyroid gland, parathyroid gland, skeletal muscle, bone marrow, peripheral nerve, spinal cord and brain).

In all cases, rats fed diets containing 1% test substance displayed a significant growth depression throughout the major part of the study. No adverse effects were apparent at lower dietary levels. The growth effect observed among animals at the 1% dietary level was attributable to the palatability of the test diet. Animal survival was not however influenced by the ingestion of the test material at any dose level. As a result, the reported NOAEL for the studies was 0.05% or 0.2% in the diet (equivalent to approximately 50 or 200 mg/kg/day), depending on the dose levels used. However, for the purposes of human hazard assessment, the reduced growth observed for the high dose group is not relevant, since this is related to exposure route. The NOAEL can therefore be considered to be >200 mg/kg/day for risk characterisation purposes.

Sterols: In a published study (Hepburn et al., 1999), phytosterol esters (PE) were added to the diet of male and female Wistar derived rats over a period of 90 days, to give concentrations of 0.16, 1.6, 3.2, and 8.1% (w/w) equivalent to phytosterol concentrations of 0.1, 1.0, 2.0, and 5.0 % (w/w) respectively. The results of this study showed there to be no clinical signs or effects on survival attributed to the administration of the test material. A NOEL of 8.1% (PE) was identified in the study, which is equivalent to a dose of 6.6 g/kg/bw/day PE or 4.1 g phytosterol/kg bw/day.

In summary,

Constituent type	Proportion in DTO (%)	NOAEL (mg/kg/day, rat)	Duration	Remarks
DTO	100	>1000	90 days	OECD 408
Fatty acids	66	≥12,500	90 days
Resin acids and neutrals	30	>200	2 year	Weight of evidence from a number of 90-day and 2-year studies
Sterols	-	≥4100	90 days

 

These data are sufficient to demonstrate that none of the constituents of DTO require classification for specific target organ toxicity following repeated oral exposure, and to allow quantitative Derived No Effect Level to be set for risk characterisation.

Justification for classification or non-classification

The available data for DTO and constituents of DTO indicate that classification for specific target organ toxicity is not required according to the criteria set out in Regulation EC No. 1272/2008.