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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Conducted for the National Toxicology Program according to robust study protocol. Detailed summary report published, but no idividual data presented (group mean data only).

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no neurological observation battery.
Principles of method if other than guideline:
Design similar to OECD 408, but did not include functional obseration battery. Additional tests were included to assess potential reproductive effects and metabolism.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Lot TA 821004-1 (Dow Chemical Company, Midland MI), purity > 99%

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Rats obtaind from Taconic Farms (Germantown, NY) and acclimatised in the facility for 12 - 14 days prior to the start of the study, approximatley 6 weeks old at first exposure. Housed in groups of 5/sex in polycarbonate cages with hardwood chip bedding. Tap water (glass bottles) and diet (NIH-07 Open Formula mash) available ad libitum. Temperature was maintained between 20.0 to 23.3 0C and relative humidity 45 to 64 %. Fluorescent lighting was on a 12 hours/day cycle and room air changes were at least 10/hour.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The test material was microencapsulated by Midwest Research Institute (Kansas City, MO) into shells composed of 80 % food-grade modified corn starch and 20 % sucrose. Mean load in each microcapsule was 50.8 + 0.3 %; analysis confirmed no additional impurities from encapsulation. There was no measurable loss of chemical (by weight) after 28 days (at 25 0C and 50 % relative humidity), and 99.1 % of initial weight was retained following 7 freeze-thaw cycles.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability studies of the dosed feed mixture containing 2.5 mg 1,1,1-trichloroethane/g diet were assessed using gas chromatography with flame ionisation detector (no further details of the method specified). Homogeneity was confirmed. Stability samples stored at room temperature in sealed containers lost 1.9, 5.3 and 7.3 % after 7, 14 and 21 days, respectively. After 1 day exposure to air and light at 26 0C in relative humidity for 1 day there was a loss of 8.9 %, with no change after 6 days.

Dose concentration was analysed periodically with gas chromatography and all dose formulations were within 10 % of the theoretical concentration (actual time points and values not specified). Analysis of actual cage samples give greater losses in concentration and the report speculates that this may have been due to moisture in the feed hoppers.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Continuous dietary exposure
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5,000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
10,000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
20,000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
40,000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
80,000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 males + 10 females per group for toxicity assessment
10 males + 10 females per group for clinical pathology investigations.
Control animals:
yes, plain diet
other: placebo microcapsules
Details on study design:
Rats were house in groups of 5/sex/treatment. NIH-07 Open Formula mash and water were available ad libitum. The frequency of diet preparation is not stated, but no prepared diets were stored for greater than 21 days in sealed containers. Treated diets were availalbe for 13 weeks.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
Animals were observed twice daily. Body weight and clinical findings were recorded weekly. Food consumption was measured twice weekly (over 3 to 4 day intervals). Blood samples for haematology and clinical chemistry were collected from the retro-orbital sinus (under carbon dioxide anaesthesia) on days 3 and 23 of exposure from an additional group of 10 male and 10 female rats; all remaining rats were sampled after 13 weeks. Twenty-four hour urine samples were collected from 5 randomly selected male rats in each of the vehicle control, 5,000, 20,000 and 80,000 ppm groups on Days 28 and 84.
Haematology samples were collected into EDTA and examined for haematocrit, haemoglobin, erythrocyte, reticulocyte; and nucleated erythrocyte counts, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, platelet count and leukocyte count and differentials.

Clinical chemistry samples were allowed to clot and the serum removed after centrifugation and examined for blood urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase and bile acids.

Urine samples were stored on ice then assessed for volume. Samples were mixed with deionised water and urine creatinine, trichloroacetic acid, and free and total tricloroethanol concentrations were measured with gas chromatography (hydrochloric acid was added for the extraction of trichloroacetic acid)
Sacrifice and pathology:
At termination animals were killed by carbon dioxide asphyxiation and subject to necropsy. The weight of the heart, right kidney, liver, lungs, right testis and thymus were recorded. Tissues were fixed in 10 % neutral buffered formalin. Complete histopathological examinations were performed on untreated controls, vehicle controls and rats exposed to 80,000 ppm. The following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, oesophagus, heart, large intestine (caecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, preputial gland, prostate gland, salivary gland, spleen, stomach (fore stomach and glandular stomach), testis (with epididymidis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder and uterus. In addition the kidneys of all rats were examined.

Other examinations:
Vaginal cytology and sperm motility were performed after 13 weeks on 10 male and 10 female rats in the vehicle control, 20,000, 40,000 and 80,000 ppm groups. Virginal lavage samples were taken for 12 days prior to sacrifice to determine oestrous cycle stage. Sperm mobility was evaluated at necropsy on slides with test yolk; 2 to 5 microscopic fields per slide were evaluated by two observers. Density, spermatogenesis were also assessed.
Statistics:
Lesion incidence was assessed using the Fisher exact test. Normally distributed data (organ and body weights) were analysed using Dunnett and Williams test. Non-parametric data (haematology, clinical chemistry, spermatid and epididymal spermatozoa data) were analysed using the multiple comparison methods of Shirley and Dunn.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
There were no treatment related effects on survival or clinical observations. The final mean body weight and body weight gain of males given the vehicle control was greater than the untreated diet control, indicating that the placebo micro-capsules had some calorific value. Food consumption was similar in all untreated and treated groups. The body weight gain of females given 20,000 ppm and males given 40,000 or 80,000 ppm was significantly lower than the untreated controls; however all final weights were within 10 % of the untreated controls.

Minimal erythrocytosis (decreased haematocrit values, haemoglobin concentration and erythrocyte counts) were noted in males and females given 10,000 ppm or greater on Days 3 and 23; in females the effect was also apparent at Week 13. These findings are consistent with a minimal relative erythrocytosis related to haemoconcentration. These changes were minimal and considered to be due to physiological processes and not directly related to the administration of 1,1,1-trichloroethane. There were no effects on male urinary volume or creatinine concentrations.

The absolute liver weight of males given 10,000 or 80,000 ppm was statistically significantly lower than the controls. However, this was no longer apparent when adjusted for body weight, was without dose response and is considered to reflect the lower treated male body weights and not an effect of treatment. In females given 80,000 ppm absolute and body weight related liver weight was lower than the controls; however in the absence of any corresponding clinical pathology or histopathological changes this is considered to be without toxicological significance. There were no other effects on organ wieght.

Urinary volume measurements and creatinine concentrations were not affected by treatment. Urinary tricloroacetic acid, free trichloroethanol and total tricloroethanol concentrations were raised in treated rats.

The epididymal spermatozoal concentration of males given 80,000 ppm was significantly less (by approximately 10 %) than the vehicle controls. Vaginal cytology parameters of exposed females were similar to controls.

Non-neoplastic kidney lesions were observed in male rats exposed to 20,000 ppm or greater; incidence and severity was dose related. Observations included renal tubule hyaline degeneration, regeneration, cast formation and chronic interstitial inflammation. These lesions are consistent with hyaline droplet nephropathy, which results from the accumulation of α2u-globulin in the renal tubules; generally this type of nephropathy is reversible after the cessation of exposure and is not considered applicable to human risk assessment.

Effect levels

Dose descriptor:
NOEL
Effect level:
10 000 ppm
Sex:
male/female
Basis for effect level:
other: based on hyaline droplet nephropathy (male rats only, 20,000 ppm and greater) and decreased mean body weight (females 20,000 ppm, males 40,000 and 80,000 ppm)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean bodyweights (BW) and bodyweight gains (BWG)a

n = 10

Untreated control

Vehicle control

1,1,1-Trichloroethane ppm (diet)

5,000

10,000

20,000

40,000

80,000

 MALES Initial BW (g)

 144 + 3

 148 + 3

146 + 3

148 + 3

149 + 3

146 + 3

147 + 3

 BWG Wk 13 (g)

 321 + 4

340 + 6* 

332 + 4

344 + 4**

336 + 4

322 + 4’

307+ 6’’

 BWG Wk 1-13 (%untreated C)

 

 

98

101

99

95

90

 FEMALES Initial BW

 107 + 1

108 +

108 +

108 +

106 +

105 +

106 +

 BWG Wk 13 (g)

 188 +

 189 + 3

194 +

189 +

178 + 3*

188 +

181 + 2’

 BWG Wk 1-13 (%untreated C)

 

 

 103

100

94

99

96

a Data obtained from page 26 in the study report.

*  Significantly different (p 0.05) from the control, Dunnett’s test

** Significantly different (p 0.01) from the control, Dunnett’s test

‘  Significantly different (p 0.05) from the control, William’s test

‘’ Significantly different (p 0.01) from the control, William’s test

 Table 2: Mean Feed Consumption (FC) and Average Dose (BWG)a

n = 10

Untreated control

Vehicle control

1,1,1-Trichloroethane ppm (diet)

5,000

10,000

20,000

40,000

80,000

MALES FC Wk 1 to 13 (g/kg/day)

58.8

58.5

57.9

60.7

59.4

59.4

60.2

Average Dose (mg/kg/day)

290

600

1,200

2,400

4,800

FEMALES FC Wk 1 to 13 (g/kg/day)

62.6

62.8

62.5

64.3

62.9

62.2

62.6

Average Dose (mg/kg/day)

310

650

1,250

2,500

5,000

a Data obtained from page 26 in the study report.

Table 3: Selected haematology findings

Untreated control

Vehicle control

1,1,1-Trichloroethan ppm (diet)

5,000

10,000

20,000

40,000

80,000

Number of MALES/group

10

10

10

10

10

10

10

Haematology Day 3

 - Haematocrit (%)  mean

43.4

42.3

43.5

43.8’

43.7’

43.9’

45.4**’’

SD

0.4

0.6

0.5

0.2

0.4

0.6

0.4

 - Haemoglobin (g/dL) mean

14.2

13.8

14.3

14.3

14.3

14.3

14.8**’’

SD

0.1

0.2

0.1

0.1

0.1

0.2

0.1

 - Erythrocytes (106/μL) mean

7.20

7.10

7.23

7.24

7.23

7.25

7.54*’’

SD

0.08

0.09

0.07

0.06

0.05

0.09

0.07

Haematology Day 23

 - Haematocrit (%) mean

48.2

47.8

48.7

49.0

49.6’

49.0

50.0**’’

SD

0.4

0.4

0.2

0.3

0.6

0.4

0.4

 - Haemoglobin (g/dL) mean

16.1

15.9

16.1

16.3’

16.4

16.4

16.6*’’

SD

0.1

0.2

0.1

0.1

0.2

0.2

0.1

 - Erythrocytes (106/μL) mean

8.37

8.28

8.49’

8.59’’

8.71*’’

8.53’

8.68*’’

SD

0.05

0.08

0.07

0.07

0.10

0.08

0.08

Number of FEMALES/group

10

10

10

10

10

10

10

Haematology Day 3

 - Haematocrit (%)  mean

43.6

43.69

43.9

44.0

44.5

44.8

44.8

SD

0.4

0.4

0.6

0.3

0.5

0.4

0.6

 - Haemoglobin (g/dL) mean

14.4

147.5

14.6

147.6

14.9

14.9*

15.1*’

SD

0.1

0.2

0.25

0.1

0.2

0.1

0.2

 - Erythrocytes (106/μL) mean

7.35

7.36

7.47

7.50

7.61

7.61’

7.72

SD

0.09

0.09

0.11

0.06

0.11

0.07’

0.11*’

Haematology Day 23

 - Haematocrit (%) mean

46.8

46.4

46.7

47.2

46.5

46.7

48.1’

SD

0.5

0.4

0.3

0.4

0.5

0.4

0.3’

 - Haemoglobin (g/dL) mean

16.0

16.0

16.0

16.0

15.9

15.9

16.4

SD

0.1

0.1

0.1

0.1

0.1

0.1

0.1

 - Erythrocytes (106/μL) mean

8.07

7.99

8.12

8.14

8.22

8.09

8.45*’’

SD

0.08

0.05

0.07

0.07

0.09

0.08

0.08

Haematology Week 13

 - Haematocrit (%) mean

45.5

45.5

45.4

45.8

47.5’

47.3**’’

49.3**’’

SD

0.3

0.3

0.3

0.3

0.8

0.4

0.4

 - Haemoglobin (g/dL) mean

15.3

15.3

15.4

15.5

15.9

15.9**’

16.4**’’

SD

0.1

0.1

0.1

0.1

0.3

0.2

0.2

 - Erythrocytes (106/μL) mean

8.59

8.48

8.46

8.52

9.11’

8.79

9.38**’’

SD

0.11

0.14

0.14

0.17

0.19

0.16

0.08

a Data obtained from pages 64 -69 in the study report.

*  Significantly different (p 0.05) from the untreated control, Dunn’s or Shirley’s test

** Significantly different (p 0.01) from the untreated control, Dunn’s or Shirley’s test

‘  Significantly different (p 0.05) from the vehicle control, Dunn’s or Shirley’s test

‘’ Significantly different (p 0.01) from the vehicle control, Dunn’s or Shirley’s test

Table 4: Incidences of Selected Neon-neoplastic Lesions in the Kidney

Untreated control

Vehicle control

1,1,1-Trichloroethane ppm (diet)

5,000

10,000

20,000

40,000

80,000

Number of MALES/group

10

10

10

10

10

10

10

Inflammation, Chronic

0

0

0

2

7**’’

10**’’

10**’’

 - mean severity

1.0

1.0

1.1

1.2

Renal Tubule, Casts

0

0

0

0

1

5*’

10**’’

- mean severity

1.0

1.0

2.0

Renal Tubule, Degeneration, Hyaline

0

0

0

0

10**’’

10**’’

10**’’

 - mean severity

1.2

1.1

2.0

Renal Tubule, Regeneration

10

10

10

10

10

10

10

 - mean severity

1.0

1.2

1.1

1.5

1.8

1.8

2.2

a Data obtained from page 30 in the study report.

*  Significantly different (p 0.05) from the untreated control, Fisher exact test

** Significantly different (p 0.01) from the untreated control, Fisher exact test

 ‘  Significantly different (p 0.05) from the vehicle control, Fisher exact test

‘’ Significantly different (p 0.01) from the vehicle control, Fisher exact test

Severity score: 1 = minimal, 2 = mild, 3 = moderate, 4 = severe

Table 5: Urinary Metabolite Data

Vehicle control

1,1,1-Trichloroethane ppm (diet)

5,000

20,000

80,000

Number of MALES/group

5

5

5

5

Day 28

- Urine creatinine (mg/dL) mean

95.0

84.0

111.0

105.0

SD

8.4

5.3

7.8

7.1

- Trichloroacetic acid (μg/mg creatinine) mean

2.39

8.05**

38.52**

86.95**

SD

0.19

0.43

4.09

9.57

- Free trichloroethanol (μg/mg creatinine) mean

0.67b

32.50*

115.59**

139.79**

SD

0.37

17.24

42.78

97.14

- Total trichloroethanol (μg/mg creatinine) mean

3.3

156.0**

629.5**

1085.7**

SD

0.2

15.3

115.5

81.3

Day 84

- Urine creatinine (mg/dL) mean

147.0

138.0

165.0

163.0

SD

2.5

6.0

13.7

25.9

- Trichloroacetic acid (μg/mg creatinine) mean

0.778b

12.874*

15.772**

73.337**

SD

0.025

1.533

1.933

15.265

- Total trichloroethanol (μg/mg creatinine) mean

2.6

136.1**

315.9**

1147.8**

SD

0.1

14.9

24.5

105.4

a Data obtained from page 69 in the study report.

*  Significantly different (p 0.05) from the control group by Shirley’s test 

** Significantly different (p 0.01) from the control group by Shirley’s test 

b n = 4

Table 6: Male Reproductive Evaluation

Vehicle control

1,1,1-Trichloroethane ppm (diet)

5,000

20,000

80,000

Number of MALES/group

10

10

10

10

Epididymal spermatozoal measurements

- Concentration (106/g cauda epididymal tissue) mean

755

713

722

677*

SD

19

18

22

15

a Data obtained from page 76 in the study report.

*  Significantly different (p 0.05) from the vehicle control group by Dunn’s test

Applicant's summary and conclusion

Conclusions:
The no-observed-adverse-effect level (NOAEL) was estimated to be 10,000 ppm for male and female rats, based on hyaline droplet nephropathy (male rats only, 20,000 ppm and greater), decreased liver weight in females (80,000 ppm only and not of toxicological significance) and decreased mean body weight (females 20,000 ppm, males 40,000 and 80,000 ppm). Based on dietary consumption, this is equivalent to 600 mg/kg/day in males and 650 mg/kg/day in females.
Executive summary:

Groups of 10 male and 10 female Fischer rats were exposed to 1,1,1trichloroethane via dietary encapsulation at feed concentrations of 5,000, 10,000, 20,000, 40,000 and 80,000 ppm for 13 weeks.  Clinical observations, body weight and food consumption were measured.  Additional animals were used to investigate effects on haematology and clinical chemistry during the study.  Additional males were used for measurement of urinary metabolites.  Limited reproductive assessment included vaginal cytology and sperm assessments.  Terminal investigations included haematology and clinical chemistry, organ weight, necropsy and histopathology.

The no-observed-adverse-effect level (NOAEL) was estimated to be 10,000 ppm for male and female rats, based on hyaline droplet nephropathy (male rats only, 20,000 ppm and greater), decreased liver weight in females (80,000 ppm only and not of toxicological significance) and decreased mean body weight (females 20,000 ppm, males 40,000 and 80,000 ppm).  Based on dietary consumption, this is equivalent to 600 mg/kg/day in males and 650 mg/kg/day in females.