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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
review article or handbook
Title:
Toxicological Evaluations - 8 - Thioglycolic acid 2-ethylhexyl ester
Author:
BG Chemie
Year:
1993
Bibliographic source:
BG Chemie, Potential Health Hazards of Existing Chemicals - Toxikologische Bewertungen Nr. 83
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
Study conducted prior to the adoption of the most recent version of this Guideline
Deviations:
yes
Remarks:
Neurobehavioural examination was not performed; weight of epididymides, thymus, spleen, brain and heart were not measured; gross pathology and histopathology were not conducted on all required tissues.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl mercaptoacetate
EC Number:
231-626-4
EC Name:
2-ethylhexyl mercaptoacetate
Cas Number:
7659-86-1
Molecular formula:
C10H20O2S
IUPAC Name:
2-ethylhexyl sulfanylacetate
Details on test material:
- Name of test material (as cited in study report): TA2-E
- Physical state: colourless liquid
- Storage condition of test material: stored in its original container, in the dark, at 2-6 °C

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac Ltd., UK
- Age at receipt:
Males: 39-45 days old
Females: 40-46 days old
- Weight at study initiation:
Males: 110 -140 g
Females: 93-111 g
- Fasting period before study: no
- Housing: individually in propylene cages
- Diet: commercial diet ad libitum
- Water: domestic tap water ad libitum
- Acclimation period:
Males: 7 days
Females: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 28 July To: 27 August 1987

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: rodent diet using a mechanical mixer
- Storage temperature of food: 2-6 °C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sample of test article/diet combination was analysed immediately following preparation to check that the concentration in test article was within +/- 5% of the intended concentration. Diets in the storage containers were re-analysed on day 7 and then, discarded. Analysis was performed using Gas liquid Chromatography.
Duration of treatment / exposure:
28 days
Frequency of treatment:
continuously in diet
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg diet
Remarks:
equivalent to 43 mg/kg bw/d
Dose / conc.:
1 000 mg/kg diet
Remarks:
equivalent to 84 mg/kg bw/d
Dose / conc.:
2 000 mg/kg diet
Remarks:
equivalent to 170 mg/kg bw/d
No. of animals per sex per dose:
5
Control animals:
yes, plain diet
other: positive control
Details on study design:
Post-exposure period: none
- Dose selection rationale: based on 14-d range-finding study results (study not available (BIBRA, 1989) described in BG Chemie (1993)):
In a preliminary study prior to the 4 week feeding study, groups of 2 male and 2 female Sprague Dawley rats were given a diet containing 500, 1000, 2000 or 4000 ppm thioglycolic acid 2 ethylhexyl ester for 14 days. The rats in the 4000 ppm group suffered weight loss over the entire period and those in the 2000 ppm group had a lower weight gain than the controls. Feed consumption in the rats receiving the 4000 ppm diet was only half that of the controls and feed intake in the rats in the 2000 ppm group was slightly lower than that of the controls in both sexes. Post mortems on the rats in the 4000 ppm group revealed poor general condition, but otherwise, no treatment related effects. It was concluded from these results that 4000 ppm thioglycolic acid 2 ethylhexyl ester in the diet was unpalatable, and a maximum of 2000 ppm was scheduled for the subsequent 4 week study (BIBRA, 1989).
Positive control:
DEHP (di-(2-ethylhexyl) phthalate) at 1.2%: to investigate peroxysome proliferation and of the appropriate marker enzymes.

Examinations

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: on three days before the start of treatment, on the day of start of treatment and twice weekly until the end of the treatment period.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated: Yes using the analysed dietary concentration of TA2-E or DEHP, and the individual values for body weight and food intake.


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: end of treatment period
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes
- How many animals: 49 (all control and tested animals except one female of the highest tested group)
- Following parameters were examined.
WBC = White blood cells
RBC = Red blood cells
Hb = Haemoglobin
HCT = Haematocrit
MCV = Mean cell volume
MCH = Mean cell haemoglobin
MCHC = Mean cell haemoglobin concentration
Platelets
N = Neutrophils
E = Eosinophils
L = Lymphocytes
M = Monocytes
PT = Prothrombin time


CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood: end of treatment period
- Animals fasted: Yes
- How many animals: 49 (all control and tested animals except one male of the highest tested group)
- Following parameters were examined.
ALKP = alkaline phosphatase
CHOL = cholesterol
Cl = chloride
GLUC = glucose
ALAT = alanine aminotransferase
K = potassium
Na = sodium
ASAT = aspartate aminotransferase
PROT = total protein
ALB = albumin
TRIG = triglycerides
UREA = urea


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Any abnormalities of the external condition and of the thoracic or abdominal viscera were noted (see table 2 for details on organ weights).
HISTOPATHOLOGY: Yes (see table 3 for details)
Other examinations:
- Liver biochemistry: whole homogenates were prepared from the liver samples from each animal. These were analysed for protein concentration and cyanide-insensitive palmitoyl-CoA oxidation. The microsomal fraction was separated and examined for protein concentration and the rate of lauric acid 11- and 12-hydroxylation.
Statistics:
The continuous variable data from the control and treated groups were compared using analysis of variance followed by least significant difference test. The control and DEHP groups were compared using a two sample or pooled t test. Incidence data from the histopathological examination were compared using Fisher's exact test.
Observations of neutral lipid in liver sections from the control and TA2-E treated groups were compared using a trend analysis (Maxwell, 1961).
In all cases a level of probability less than 0.05 was taken to indicate statistical significance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
No abnormalities of condition or behaviour were seen in female rats given test substance or DEHP ((di-(2-ethylhexyl) phthalate) at 1.2%). Swelling and/or staining under one eye were observed early in the study in two male rats from the 0.05% tested group, one male rat from the 0.1% tested group and one male rat from the 0.2% tested group.
One of the affected rats in the 0.05% TA2-E group was replaced with a spare animal; the eye condition cleared up in the other three rats by study day 14. It is not considered that these isolated observations are related to treatment.


BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant differences between the bodyweights of the male control and treated groups, although during the treatment period the bodyweights of treated rats were slightly lower than those of the control rats. The weights of male rats given 1.2% DEHP were also slightly lower than controls throughout the study, but the differences again were not statistically significant.
The mean bodyweights of female rats treated with test substance or 1.2% DEHP did not differ significantly from the control group, and the treated rats gained as much weight as the control animals during the course of the study.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no statistically significant differences between the food intakes of male control rats and male rats treated with the three dose levels of test substance or 1.2% DEHP.
Female rats treated with test substance consistently consumed more diet than the control group throughout the study. These differences were statistically significant for female rats treated with 0.05% test substance on study days 20-24 and 24-27 respectively, and for animals given 0.1% test substance on study days 20-24. No dose-related effects were observed.

The food intakes of female rats treated with 1.2% DEHP did not differ statistically from the control group.


HAEMATOLOGY
The mean cell haemoglobin for male rats treated with 0.2% test substance was statistically significantly lower than the controls.
The white blood cell and lymphocyte counts for TA2-E treated male rats were lower than the controls, and these differences were statistically significant for the 0.1% and 0.2% test substance dose groups. Prothrombin times for male rats administered 0.05% and 0.1% test substance were statistically higher than the control group.
Female rats treated with 0.2% test substance had statistically higher haematocrit values, mean cell volumes and platelet counts than the control animals.

According to authors, statistical changes were not found to be toxicologically relevant, as findings in males were not elicited in females and no compound- and dose-related effects were demonstrated.

Comparison of the haematological measurements for the control group and DEHP-treated rats showed some statistically significant differences. In the female rats the erythrocyte parameters: haemoglobin concentration, mean cell volume, mean cell haemoglobin and mean cell haemoglobin concentration, were all statistically different for the control and DEHP-treated animals and the platelet counts were higher.
In male rats, prothrombin times for DEHP-treated animals were higher than the controls.


CLINICAL CHEMISTRY
There were no statistically significant differences between control and test substance treated female rats, in the three dose groups, for any of the serum chemical measurements.
For male rats the only statistically significant difference between control and test substance treated animals was an increase in aspartate aminotransferase activity in the 0.2% test substance group.

A few not significant differences were seen between control and DEHP-treated rats.


ORGAN WEIGHTS
Male rats treated with the test substance had increased kiney weight when compared to controls. Relative kidney weight to bodyweight increases at 0.05% and 0.2% were statistically significant. Increases but no statistically significant changes were elicited for absolute kidney weight.
Female rats treated with 0.1% test substance had significantly higher absolute and relative kidney weights than the control animals. The absolute kidney weights of female rats treated with 0.05% of test substance were also statistically significantly higher than those of the control females.
Male liver weights were not affected by treatment with test substance. In the test substance treated female groups, the only statistically significant effect on liver weights was an increase in the relative liver weights of animals given 0.1% test substance, compared with controls.

The absolute and relative liver weights in both sexes given 1.2% DEHP were statistically significantly higher than those of the control group. The relative kidney weights from both sexes in the DEHP group were also statistically significantly higher than those of the controls. In the female rats this difference was also apparent from the statistical analysis of the absolute kidney weights.

Changes in kidney weights observed in some test substance treated animals were not dose related. Increases of the liver weight were confirmed by biochemical examination (induction of lauric acid hydroxylation) in 1.2% DEHP treated animals but not in test substance treated animals. In DEHP-treated animals, the livers were enlarged, and palmitoyl-CoA oxidation and lauric acid 11¬and particularly 12-hydroxylase activities were increased. This pattern of observations is typical of those chemicals which cause hepatic peroxisome proliferation in the rat.


GROSS PATHOLOGY
A few animals from all groups showed minor changes in the appearance of their liver, kidneys or lungs. The pattern of incidence was unrelated to treatment with either test substance or DEHP.


HISTOPATHOLOGY: NON-NEOPLASTIC

Light microscopic examination:
Histopathological examination of the kidneys from female rats treated with the three dose-levels of test substance showed a statistically significant incidence of nephrocalcinosis. This finding is not unusual in this sex, and is not considered to be related to treatment with test substance. Nephrocalcinosis was absent in female rats treated with DEHP, and was not seen in male rats treated with either test substance or DEHP.
Examination of the lungs from 2 animals (one control male and one female treated at the lowest dose level) showed interstitial pneumonitis and perivascular cuffing. Microgranuloma were also seen in the lungs from one of these animals.
Haematoxylin and eosin stained liver sections from male and female rats treated with test substance did not show any significant histological changes compared with the control groups.
Statistical analysis showed no significant trend for the reduction in periportal fat with increasing dose of test substance.
Treatment with 1.2% DEHP produced a reduction in periportal fat in the livers from both sexes. A statistically significant incidence of reduced cytoplasmic basophilia was also observed in this group of animals.

Electron microscopic examination:
When compared to control, microscopic examination of liver peroxisomes of animals treated with 0.2% did not reveal any major changes that could be attributed to treatment. Only a variation in the number of peroxisomes per cell was indicated for treated male rats.
In both male and female rats, administration of 1.2% DEHP to female rats produced a moderate increase in the size and number of peroxisomes per cell, evident in both portal and centrilobular areas. Peroxisomes showed a variation in size with many of the larger organelles possessing irregular profiles and no dense core.


OTHER FINDINGS

Liver Biochemistry:
Hepatic protein concentrations were slightly higher than controls in male and female rats treated with the two top doses (0.1% and 0.2%), though the differences were not statistically significant. Treatment with test substance did not produce any statistically significant increases in cyanide-insensitive palmitoyl-CoA oxidation or lauric acid 11- and 12-hydroxylation in male or female rats.
There were no significant changes in microsomal protein concentrations of test substance treated male and female rats compared with the control group.

Hepatic protein concentrations were higher than the controls for DEHP-treated male and female rats, and the differences were statistically significant for female rats. The microsomal protein concentrations of male and female rats treated with DEHP were similar to the controls, but the palmitoyl-CoA oxidation and lauric acid 11- and 12-hydroxylation were markedly higher than the control group. In both sexes treatment with 1.2% DEHP gave a greater induction of lauric acid 12-hydroxylation than lauric acid 11-hydroxylation.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 170 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Administration of the substance in the diet was not found to produce any treatment-related effects at the concentrations tested.
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Haematology and clinical chemistry – Statistical changes and corresponding values in the opposite sex

Exposure Group

Control

43 mg/kg/d

84 mg/kg/d

170 mg/kg/d

Males

Haematology

WBC (10E3/µL)

10.68 +/- 2.1

10.48 +/- 0.81

8.82 +/- 0.95*

8.56 +/- 0.76*

MCH (pg)

25.47 +/- 0.85

24.40 +/- 0.35

24.68 +/- 0.54

23.70 +/- 1.3**

Lymphocytes (10E3/µL)

8.21 +/- 2.0

7.94 +/- 0.82

6.29 +/- 0.66*

6.55 +/- 0.32*

Prothrombin time (sec)

9.38 +/- 0.06

9.97 +/- 0.48*

10.04 +/- 0.27**

9.66 +/- 0.31

HCT (% of total blood)

39.52 +/- 1.6

41.72 +/- 0.9

41.46 +/- 1.1

42.46 +/- 2.6

MCV (µmE3)

58.8 +/- 0.5

59.0 +/- 0.7

58.2 +/- 0.8

58.6 +/- 0.9

Platelets (10E3/µL)

918.5 +/- 74.9

970.4 +/- 25.4

932.6 +/- 42.1

971.6 +/- 57.5

Clinical Chemistry

ASAT (International units)

123 +/- 19.3

111 +/- 23.8

106 +/- 14.4

155 +/- 24.5*

Females

Haematology

HCT (% of total blood)

42.22 +/- 2.2

44.40 +/- 2.6

44.42 +/- 0.84

47.82 +/- 2.6**

MCV (µmE3)

60.6 +/- 0.89

61.6 +/- 1.1

61.6 +/- 0.55

64.3 +/- 1.9***

Platelets (10E3/µL)

883.6 +/- 50.5

939.2 +/- 79.1

938.8 +/- 42.5

1046.5 +/- 100.7**

WBC (10E3/µL)

8.6 +/- 0.7

8.1 +/- 1.9

6.9 +/- 0.5

8.9 +/- 0.5

MCH (pg)

26.0 +/- 1.3

25.8 +/- 1.1

25.6 +/- 0.6

24.7 +/- 1.8

Lymphocytes (10E3/µL)

5.91 +/- 1.1

5.75 +/- 1.2

4.91 +/- 0.4

6.27 +/- 0.2

Prothrombin time (sec)

8.56 +/- 0.34

8.42 +/- 0.14

8.75 +/- 0.69

8.89 +/- 0.67

Clinical Chemistry

ASAT (International units)

100 +/- 7.0

101 +/- 5.2

104 +/- 9.0

97 +/- 10.5

*Statistically significant difference from control group (p0.05) using least significant difference test.

**Statistically significant difference from control group (p0.01) using least significant difference test.

***Statistically significant difference from control group (p0.001) using least significant difference test.

Organ Weights – Statistical changes

Exposure Group

Control

43 mg/kg/d

84 mg/kg/d

170 mg/kg/d

Males

Relative organ Weight (g) to bodyweight

Kidney

0.658 +/- 0.028

0.688 +/- 0.026*

0.656 +/- 0.005

0.699 +/- 0.017**

Females

Absolute organ Weight (g)

Kidney

0.979 +/- 0.040

1.054 +/- 0.059*

1.070 +/- 0.038*

1.009 +/- 0.061

Relative organ Weight (g) to bodyweight

Liver

2.73 +/- 0.10

2.80 +/- 0.09

2.92 +/- 0.12**

2.75 +/- 0.07

Kidney

0.689 +/- 0.024

0.712 +/- 0.023

0.765 +/- 0.054**

0.713 +/- 0.02

*Statistically significant difference from control group (p<0.05) using least significant difference test.

**Statistically significant difference from control group (p<0.01) using least significant difference test.

Table 6 Incidence of histopathological findings – Statistical changes

Exposure Group

Control

43 mg/kg/d

84 mg/kg/d

170 mg/kg/d

Females

Kidney

Nephrocalcinosis

1/5

5/5*

5/5*

5/5*

*Statistically significant difference from control group (p<0.05) using Fisher’s exact test.

**Statistically significant difference from control group (p<0.01) using least significant difference test.

Applicant's summary and conclusion

Conclusions:
In this 28-day oral study, in which rats were given 43, 84 and 170 mg/kg bw/d in diet, the NOAEL was determined to be higher than 170 mg/kg bw/d, as no significant adverse effect could be attributed to test substance treatment.
Executive summary:

Groups of male and female rats were exposed to EHTG in a feeding study at concentrations of 0, 0.05, 0.1 and 0.2% for a period of 28 days. One group of 5 males and 5 females was treated with di-(2-etlhexyl)phthalate (DEHP) as positive control for peroxisome proliferation. Clinical observations were performed daily and weekly detailed examinations were made. Body weights and food intake was measured on days 0 and 3, and then twice weekly. Macroscopic exam, organ weights, haematology and blood chemistry were performed at the end of the treatment period. Microscopic examination of selected organs was performed for control and high EHTG dose groups. Electronic microscopy was used for examination of the liver from control and high EHTG dose groups. The actual dose received by sex was as follows:

DIETARY LEVEL

MALES

FEMALES

%

(mg/kg/day)

(mg/kg/day)

0.05

42

45

0.1

82

87

0.2

168

173

There were no statistically significant differences between the bodyweights of the control and EHTG treated groups. There were no statistically significant differences between the food intakes of male control rats and male rats treated with the three dose levels of EHTG or 1.2 % DEHP. Female rats treated with EHTG consistently consumed more diet than the control group throughout the study. These differences were statistically significant for female rats treated with 0.05 % EHTG on study days 20-24 and 24-27 respectively, and for animals given 0.1 % EHTG on study days 20-24. The food intakes of female rats treated with 1.2 % DEHP did not differ statistically from the control group. No abnormalities of condition or behavior were seen in female rats given EHTG. The white blood cell and lymphocyte counts for EHTG treated male rats were lower than the controls, and these differences were statistically significant for the 0.1 % and 0.2 % EHTG dose groups. The mean cell haemoglobin for male rats treated with 0.2 % EHTG was statistically significantly lower than the controls. Female rats treated with 0.2% EHTG had statistically higher haematocrit values, mean cell volumes and platelet counts than the control animals. There were no statistically significant differences between control and EHTG treated female rats, in the three dose groups, for any of the serum chemical measurements. For male rats the only statistically significant difference between control and EHTG treated animals was an increase in aspartate aminotransferase activity in the 0.2 % EHTG group. There were no deaths. A few animals from all groups showed minor changes in the appearance of their liver, kidneys or lungs. The pattern of incidence was unrelated to treatment.Male rats administered 0.05 % and 0.2% EHTG had statistically significantly higher relativekidney weights than the controls, but no effect on absolute kidney weights. Female rats treated with EHTG had significantly higher absolute (at 0.1 % and 0.05 % of EHTG) and relative kidney weights (at 0.1% EHTG) than the control animals. Hepatic protein concentrations were slightly higher than controls in male and female rats treated with the two top doses of EHTG (0.1 % and 0.2 %), though the differences were not statistically significant. Treatment with EHTG did not produce any statistically significant increases in cyanide-insensitive palmitoyl-CoA oxidation or lauric acid 11- and 12-hydroxylation in male or female rats. There were no significant changes in microsomal protein concentrations of EHTG treated male and female rats compared with the control group. EHTG in the diet of rats for 28 days does not lead to a proliferation of hepatic peroxisomes, and does not produce any treatment-related effects. The NOAEL was 0.2 % EHTG in the diet (168 mg/kg bw for males; 173 mg/kg bw for females).