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Diss Factsheets

Administrative data

Description of key information

Repeated dose toxicity studies in laboratory animals identified the liver, kidneys and nasal cavity as the target organs for the toxic potential of chloroform. The lowest LOAEL for hepatotoxicity following repeated oral exposure to chloroform was 15 mg/kg bw/day reported from a 7-year study in dogs. A NOAEL for the mentioned target organs of 34 mg/kg bw/day was determined in female F-344 rats receiving chloroform by oral gavage for three weeks.

The NOAEC for renal lesions, induction of cell proliferation and tumour formation in the kidneys following repeated inhalation exposure of male mice to chloroform vapours for 90 days was 25 mg/m3 (exposure for 5 days a week) or 50 mg/m3 (exposure for 7 days a week).  Distinct nasal lesions in the form of a generalised atrophy of ethmoid turbinates were observed in male and female F-344 rats exposed to 50 mg/m3 for 90 days. In another key 90 -day study in mice, the NOAEC was 10 mg/m3 as changes were observed at the next higher level of 49 mg/m3. A key 90 -day study in rats resulted in a NOAEC of 49 mg/m3.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
animals were exposed 5 d/wk for 3 wks
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, North Carolina, USA)
- Age at study initiation: 9 weeks
- Weight at study initiation: 139.5 +/- 5 g
- Fasting period before study: no data
- Housing: rats were housed two or three per cage in polystyrene shoe-box cages with cellulose bedding (ALPHA-dri, Shepherd Specialty Papers, Kalamazoo, USA), and filter top lids
- Diet (e.g. ad libitum): NIH-07 rodent chow (Ziegler Bros, Gardener, Pennsylvania, USA) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Rats received chloroform dissolved in corn oil at doses of 0, 34, 100, 200 and 400 mg/kg/day in a constant dosing volume of 2 mL/kg administered by oral gavage. Each dose concentration of chloroform was prepared fresh on the morning that it was dispensed. All doses were administered between 8.00 and 10.00 hr to minimise diurnal variation.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
4 consecutive days or 3 weeks
Frequency of treatment:
Daily in the 4-day study and one administration per day on five days per week in the 3-week study
Dose / conc.:
34 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 rats per dose group
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
no data, but regular observations were performed during the testing period
Sacrifice and pathology:
At autopsy, rats were anaesthetised with sodium pentobarbital and the kidneys perfused in situ. Following a 3-min perfusion, the entire livers and kidneys were removed. Livers were weighed, examined macroscopically, and longitudinal 3-4 mm mid-sections of the left, median and right anterior hepatic lobes taken. The right and left kidneys were examined grossly and a 3-4 mm mid-saggital section of the left kidney and a 3-4 mm midtransverse section of the right kidney were taken. A 3-4 mm section of the duodenum was also included to confirm the systemic distribution and nuclear incorporation of bromodeoxyuridine (BrdU). Nasal cavities were flushed with neutral buffered 10 % formalin through the trachea; excess soft tissue was removed from the head, the lower jaw was removed, and the head was subsequently immersed in fresh fixative for no less than 48 hours and then decalcified in 5 % formic acid with ion exchange resin. Uniform blocks were cut at six levels of the nose to include all major epithelial types and blocks processed to paraffin. Tissue sections were cut at 4-5 micrometre thick. Serial sections were stained with haematoxylin and eosin or left unstained for BrdU immunohistochemistry.
Other examinations:
The labelling index (percentage of nuclei in S-phase) was evaluated in rats by bromodeoxyuridine (BrdU) immunohistochemistry. On the Monday afternoon preceding a Friday autopsy, osmotic pumps containing 20 mg/mL BrdU were aseptically implanted sc over the thoracumbular area using isofluorane anaesthesia. Autopsies were conducted on Friday mornings; consequently, rats received BrdU for about 3.5 days. BrdU-labelled tissues were mounted on "ProbOn Plus" slides (Fisher Scientific, Pittsburg, Pennsylvania, USA) to ensure adhesion during processing. The immunohistochemical detection of BrdU-labelled cells was done as described by Elridge et al. (1990, Carcinogenesis 11, 2245-2251). Cells that had incorporated BrdU were identified by the red pigment within the nuclei. Computer-generated random fields were used for scoring BrdU-labelled nuclei in the liver and kidney by light microscopy. In the liver tissue sections, at least 1000 hepatocellular nuclei in the left lobe were counted. For kidney tissue sections, at least 1000 proximal tubular cells in the cortical region were scored, as well as 1000 epithelial cells in the outer stripe of the medulla, 1000 epithelial cells in the inner stripe of the outer medulla and 1000 epithelial cells in the inner medulla. The labelling index, representing the percentage of nuclei in S-phase, was calculated by dividing the number of labelled nuclei by the total number of nuclei counted (expressed in %). For the nasal passages, quantitative studies of cell replication were confined to the base of the dorsal scroll of the first endoturbinate. These studies used the unit length labelling index method as modified by Méry et al. (1994, Toxicology and Applied Pharmacology 125, 214-227) for investigation of nasal lesions.
Statistics:
The Williams test was used to determine significant differences in liver and body weights and labelling index between treatment and control means. The Williams test is designed to establish the lowest dose that produces a response that is significantly elevated over the control in a dose-response study.
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):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mild degenerative centrilobular changes and dose-dependent increases in the hepatocyte labelling index (LI ) were observed after administration of 100 mg or more chloroform/kg/day. Rats given 200 or 400 mg/kg/day for 4 days or 3 wk had degeneration and necrosis of the proximal tubules of the renal cortex. Regenerating epithelium lining proximal tubules was seen histologically and as an increase in LI. Dose-dependent increases in LI were observed in the kidneys at doses of 100 mg or more chloroform/kg/day at both 4 days and 3 wk. Two distinct treatment-induced responses were observed in specific regions of the olfactory mucosa lining the ethmoid region of the nose. A peripheral lesion was seen at all doses used and included new bone formation, periosteal hypercellularity and increased cell replication. A central lesion was seen at doses of 100 mg or more chloroform/kg/day and was characterized by degeneration of the olfactory epithelium and superficial Bowman's glands. These observations define the dose-response relationships for the liver, kidneys and nasal passages as target organs for chloroform administered by gavage in the female F-344 rat.
Dose descriptor:
NOAEL
Effect level:
34 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: changes in liver and kidneys at the next higher level of 100 mg/kg bw/day
Critical effects observed:
not specified

Table 1: Chloroform-induced proliferation in the liver of female F-344 rats

Dose (mg/kg/day)

Labelling index in hepatocytes after:

4 days

3 weeks

0

1.6 ± 1.1

0.6 ± 0.5

34

1.0 ± 0.6

0.8 ± 0.4

100

6.0 ± 2.5 *

2.7 ± 1.5

200

11.7 ± 3.1 *

14.0 ± 9.0 *

400

14.0 ± 5.7 *

11.8 ± 15.9 *

Values are means ± SD (n = 5 rats); Asterisks indicate significant differences from the control (p < 0.05; William’s test)

Table 2: Chloroform-induced cell proliferation in the kidneys of female F-344 rats

Duration of administration

Dose (mg/kg/day)

Labelling index in:

Cortex

Outer stripe, outer medulla

Inner stripe, outer medulla

Inner medulla

4 days

0

2.1 ± 0.6

2.9 ± 0.7

1.4 ± 0.7

0.4 ± 0.4

34

2.4 ± 0.8

2.1 ± 0.9

2.0 ± 1.3

0.8 ± 0.7

100

3.2 ± 0.7

1.7 ± 0.5

1.0 ± 0.4

0.6 ± 0.3

200

17.7 ± 12.2 *

9.1 ± 16.0

6.6 ± 12.1

2.3 ± 4.3

400

41.0 ± 5.3 *

31.1 ± 2.2 *

27.3 ± 4.1 *

3.2 ± 1.8 *

3 weeks

0

1.3 ± 1.0

1.2 ± 0.8

1.2 ± 0.7

0.4 ± 0.3

34

1.5 ± 0.3

1.0 ± 0.5

1.1 ± 0.6

0.2 ± 0.3

100

22.4 ± 20.9 *

4.9 ± 6.8

3.3 ± 3.9

0.6 ± 0.6

200

33.8 ± 20.9 *

10.4 ± 12.0 *

6.1 ± 7.1

5.1 ± 7.5

400

13.5 ± 6.9 *

5.7 ± 2.4 *

2.0 ± 1.4

0.5 ± 0.6

Values are means ± SD (n = 5 rats); Asterisks indicate significant differences from the control (p < 0.05; William’s test)

Table 3: Severity of chloroform-induced nasal lesions (mean subjective score) in femal F-344 rats

Dose (mg/kg/day)

Peripheral olfactory mucosal lesion a)

Central olfactory mucosal lesion b)

4 days

3 weeks

4 days

3 weeks

0

0

0

0

0

34

1.2

0.4

0.2

0

100

1.8

1.7

0.4

0.8

200

1.6

2.0

0.8

1.2

400

1.8

3.0

2.8

1.6

a) Periosteal hyperplasia, new bone formation, and loss of adjacent Bowman’s glands. Grading system for the turbinate bones: 0 = normal, 1 = uneven boundary of nasal bone, 2 = uneven boundary of nasal bone with slight new bone formation, 3 = moderate new bone formation, 4 = severe bone enlargement. Standard deviations are not presented owing to the subjective nature of the data and the consistent intragroup scores.

b) Degeneration of olfactory epithelium and superficial Bowman’s glands. Scoring system used for degeneration of the olfactory epithelium of the dorsal medial region of the nose: 0 = normal, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe.

Table 4: Chloroform-induced cell proliferation in the nasal turbinates of female F-344 rats.

Dose (mg/kg/day)

ULLI a)

4 days

3 weeks

0

15 ± 4

16 ± 3

34

145 ± 97 *

24 ± 9

100

306 ± 48 *

61 ± 10 *

200

321 ± 19 *

63 ± 5 *

400

377 ± 121 *

63 ± 17 *

a) Unit length labelling index of cells in the lamina propria of the proximal portion of the dorsal scroll of the first endoturbinate expressed as labelled nuclei per 0.25 mm bone. Values are means ± SD. Asterisks indicate significant differences from the control (p < 0.05, William’s test).

Conclusions:
The LOAEL for oral exposure to chloroform dissolved in corn oil administered by gavage in a subacute study using female F-344 rats was 34 mg/kg body weight/day after exposure for 4 consecutive days; the NOAEL was 34 mg/kg body weight/day after exposure for three weeks. The LOAEL was based on observations of lesions and cell proliferation in the olfactory epithelium and changes in the nasal passages.
Executive summary:

A study on the sub-acute toxicity of chloroform was carried out using female F-344 rats exposed by oral gavage to graded doses of chloroform dissolved in corn oil for 3 consecutive weeks on 5 days per week. The study was comparable to a guideline study according to method B.7 of the European Commission with acceptable restrictions. Liver, kidneys and nasal passages were identified as the target organs for chloroform administered by oral gavage. Mild degenerative centrilobular changes and dose-dependent increase in the hepatocyte labelling index (% of cells in S-phase), dose-dependent increase in labelling index in the kidneys, lesions and cell proliferation in the olfactory epithelium and changes in the nasal passages were observed at 100 mg/kg body weight/day of chloroform after 3 weeks of exposure. The NOAEL for systemic effects was 34 mg/kg body weight/day based on the finding of lesions and increased cell replication in the liver and kidneys at higher dose levels observed after repeated oral doses administered for 3 weeks. Local effects in the nasal passage were seen after 4 days of exposure at a dose of 34 mg/kg/day.

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
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
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
Deviations:
not specified
GLP compliance:
no
Remarks:
did not exist at the time
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
Pure bread dogs, initially 18 to 24 weeks old, were housed singly in kennels. All the dogs were clinically examined and inoculated against distemper, canine hepatitis and leptospirosis as well as being dose with piperazine adipate as an anthelmintic. Inoculatin and anthelmintic treatment were repeated annually. The dogs were fed weighed amounts (200 g) of a dry diet at fixed times twice daily. When new food was offered, any residue from the previous meal was removed. From week 300, the daily food ration of dogs considered to be obese was reduced from 400 to 300 g. Water was freely available at all times and fresh milk (200 mL) offered to each dog on weekday mornings for the first 6 months.
Route of administration:
oral: capsule
Vehicle:
other: toothpaste base (including peppermint oil and eucalyptol) given orally by gelatin capsule
Details on oral exposure:
Dogs received a dose of chloroform contained in tooth paste for 7 days per week during the main study.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
7.5 years
Frequency of treatment:
7 days per week
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
nominal in capsule
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
nominal in capsule
No. of animals per sex per dose:
8 males and 8 females in the 30 and 15 mg/kg body weight dose groups and in untreated and alternative non-chloroform toothpaste groups; 16 males and 16 females in the 0 mg/kg bw dose group
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
From observations made in preliminary studies and to ensure that the upper dose level would reach the threshold for early toxic effects but with many dogs as possible remaining alive and well for at least 7 years
Observations and examinations performed and frequency:
Clinical signs: daily; food consumption: daily; water consumption: intermittently; body weight: once per week; opthalmoscopy: 3-month intervals; thorough clinical examination: at least twice yearly; comprehensive range of laboratory investigations: during pre-treatment period, after 6, 13 weeks, 6.9 and 12 months, then every 6 months; serum enzyme studies: in the later stages of the treatment.
Sacrifice and pathology:
Between weeks 395 and 399, each dog still alive received an intravenous injection of sodium pentobarbitone; immediately after killing or as soon as possible in the case of animals found dead, full macroscopic examination was made of all tissues and any abnormalities were noted; principal organs were removed and weighed; small portions of these and a wide range of other tissues along with portions of any lump or tumour were placed in fixative; sections were stained with haemotoxylin and eosin; examination, by electron microscopy, was conducted on liver and kidney sections
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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
See tables below. Chronic effects related to chloroform exposure included a significantly increased level of alanine aminotransferase level in the high dose group throughout the study and in the lower-dose group during the later phase of the study.
Four out of 8 females treated at 30 mg/kg bw showed hepatic nodules but these were also seen in 3/12 vehicle controls. Aggregations of vacuolated histiocytes "fatty cysts" were observed in the liver of several animals, controls included; however, the severity in treated animals was generally moderate/marked whereas the severity in controls was generally scored as occasional/minimal.
Dose descriptor:
LOAEL
Effect level:
15 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: increased serum ALAT and increased incidence and severity of "fatty cysts" in the liver
Critical effects observed:
not specified

Table 1: Group mean values in biochemical analyses of different groups treated with chloroform, untreated control groups and groups receiving alternative toothpaste (male and female dogs combined)

Treatment (mg/kg/d)

30

15

Vehicle control

Untreated

Alternative toothpaste

No. of dogs

15

15

27

12

15

Week

374 a)

395 b)

374

395

374

395

374

395

374

395

Urea (mg %)

30

28

32

26

31

31

33

27

34

29

Glucose (mg %)

99

99

100

100

101

98

99

95

102

98

Total serum proteins (g %)

5.9

6.1

6.1

6.2

6.3

6.0

6.3

6.0

6.4

6.1

Albumin

2.9

2.8

3.0

2.8

3.1

2.8

3.2

2.9

3.2

2.9

alpha1 globulin

0.3

0.3

0.3

0.3

0.3

0.4

0.3

0.3

0.3

0.3

alpha2 globulin

0.7

0.8

0.7

0.8

0.7

0.8

0.7

0.7

0.7

0.8

beta globulin

1.6

1.7

1.7

1.7

1.7

1.6

1.6

1.6

1.7

1.6

gamma globulin

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.4

0.6

0.4

A/G ratio

0.98

0.86

0.98

0.87

0.99

0.91

1.04

0.97

1.00

0.94

SAP (KA units)

30

26

20

17

14

16

12

14

10

13

SGPT mU/mL

102

111

66

48

51

128

50

56

57

87

SGOT mU/mL

33

33

27

29

25

32

22

32

25

31

LAP (GR units)

90

72

82

62

73

54

81

56

82

60

Bilirubin (mg %)

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

Erythrocyte cholinesterase (delta pH/h)

0.73

0.73

0.78

0.81

0.77

0.81

0.77

0.82

0.65

0.71

Plasma (delat pH/h)

1.08

0.91

1.00

0.88

1.00

0.87

1.02

0.86

1.05

0.91

gamma GT (mU/mL)

3.6

3.3

2.9

2.5

2.9

2.1

2.5

1.7

2.8

1.8

GDH (mU/mL)

7.2

5.3

6.5

3.9

4.9

4.0

3.9

3.0

4.9

3.0

ICD (B&B units)

11.8

11.4

11.2

10.7

9.9

12.9

9.2

11.7

10.6

12.9

a) end of treatment; b) end of recovery period; A/G: Albumin/Globulin; SAP: Serum Amyloid P component; SGPT: Serum Glutamic Pyruvic Transaminase (also Alanine Aminotransferase); SGOT: Serum Glutamic Oxaloacetic Transaminase (also Aspartate Transaminase); gamma GT (Gamma Glutamyl Transferase); GDH: Glutamate Dehydrogenase; ICD: Isocitrate Dehydrogenase

Table 2: Changes of alanine aminotransferase (ALAT) levels throughout the main study

Treatment (mg/kg/d)

Group mean ALAT (mU/mL)

Pre-reatment

Posttreatment (weeks)

6

13

26

39

52

78

104

130

156

182

208

234

260

286

312

338

364

372

14

19

30 mg/kg/d

24

34 a)

37 b)

58 c)

63 c)

52 c)

73 c)

64 c)

51 c)

76 c)

108 c)

91 c)

80 c)

147 c)

138 c)

128 c)

134 c)

104 c)

102 c)

105 c)

111

15 mg/kg/d

22

29

30

33

39

32

43

45

34 b)

46 b)

61 b)

55 b)

55 a)

95 c)

93 b)

89 c)

73 a)

79 a)

66

53

48

Vehicle control

22

29

29

30

39

29

35

40

22

30

33

40

34

33

65

47

49

50

51

56

128

Alternative toothpaste

28

31

31

33

40

28

37

36

21

30

34

36

35

50

47

52

47

59

57

48

87

Untreated

24

30

30

30

38

27

37

37

21

29

33

30

33

32

48

50

44

50

50

53

56

a) comparison with untreated group; p 0.05; b) comparison with untreated group; p 0.01; c) comparison with untreated group; p 0.001

Table 3: Liver changes (nodules of altered hepatocytes and “fatty cysts”)

Treatment (mg/kg/d)

Sex

No. of dogs examined histologically at end of experiment

No. with nodules

No. with “fatty cysts”

Occasional or minimal

Moderate or marked

30

Male

7

0

1

6

Female

8

4

0

7

15

Male

7

1

0

6

Female

8

1

2

3

Vehicle control

Male

15

0

7

1

Female

12

3

3

0

Untreated

Male

7

1

2

0

Female

5

1

1

0

Alternative toothpaste

Male

8

0

2

0

Female

7

1

0

0
Conclusions:
This long-term study investigating chronic effects of chloroform applied orally to dogs provided a LOAEL value of 15 mg/kg body weight/day.
Executive summary:

A combined repeated dose and carcinogenicity study was carried out with chloroform orally administered to male and female Beagle dogs over a period of more than 7 years. The study was comparable to the principles set out in the testing guideline for method B.33 suggested by the European Commission with minor restrictions. The chloroform was contained in toothpaste and administered in form of a gelatine capsule. Chronic effects related to chloroform exposure included a significantly increased level of alanine aminotransferase level in the high dose group throughout the study and in the lower-dose group during the later phase of the study and increased incidence of aggregations of vacuolated histiocytes forming "fatty cysts" in the liver of treated dogs.

The LOAEL value found in this study was 15 mg/kg body weight/day established for female and male dogs together.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
15 mg/kg bw/day
Study duration:
chronic
Species:
dog
Organ:
kidney
liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
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
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Seven-week old male and female B6C3F1 mice obtained from Charles River Breeding Laboratories, Inc., Raleigh, North Carolina, USA; housed one per cage in 8-m3 stainless steel and glass inhalation chambers; separate chambers for each exposure concentration; acclimation for 2 weeks; chambers maintained on a 12-hours light/12-hours dark cycle, NIH-07 rodent chow (Ziegler Bros., Gardener; Pennsylvania), and deionised filtered tap water available ad libitum; temperature at 22.2 +/- 2 °C and 50 +/- 10 % relative humidity, continuous flow of HEPA and charcoal-filtered air at flow rates of 2000 litre/min
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
Exposure atmosphere was generated by a vaporisation technique; Nitrogen, metered by a mass flow controller, was admitted into the chloroform storage vessel through a dip tube; the chloroform-containing nitrogen gas flowed into the supply air duct for the exposure chamber; in the 2 and 10 ppm chambers, nitrogen was used to pressurise the vessel to 5 psi and carry the chloroform into the chamber through a mass flow controller
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of chloroform were monitored using a Miran 1A infrared gas analyser (see Table 1)
Duration of treatment / exposure:
see Table 2
Frequency of treatment:
6 hours on 5 days/week or 7 days/week
Dose / conc.:
1.47 mg/m³ air
Remarks:
0.3 ppm (target concentration)
Dose / conc.:
9.8 mg/m³ air
Remarks:
2 ppm (target concentration)
Dose / conc.:
49 mg/m³ air
Remarks:
10 ppm (target concentration)
Dose / conc.:
147 mg/m³ air
Remarks:
30 ppm (target concentration)
Dose / conc.:
441 mg/m³ air
Remarks:
90 ppm (target concentration)
No. of animals per sex per dose:
ranging from 10 to 60, see Table 2
Control animals:
yes
Sacrifice and pathology:
Mice in unlabelled group were weighed and killed subsequently; livers and kidneys were removed, weighed and examined macroscopically, then slides were prepared from the tissues; in rats exposed for 3 or 13 weeks, a complete tissue screen was collected including adrenals, brain, cecum, cervix, colon, duodenum, ear canal, esophagus, eye with haderian gland, femoral-tibial joint, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mesenteric lymph nodes, ovaries, pancreas, parathyroid gland, ribs, prostate, salivary gland, skin with mammary gland, sciatic nerve, seminal vesicles, spinal cord, sternum, stomach, spleen, testes, thigh muscle, thymus, thyroid, trachea, urinary bladder, uterus, vagina and vertebrae.
Other examinations:
Nasal passages were examined as well
Statistics:
The Williams test was used to determine significant differences in organ weights, body weights and labelling index between treatment and control groups; the student's t-test was used to determine statistical differences in body weight and organ weights and labelling index between the exposure groups of 7 and 5 days/week and between exposure groups of 13-week continuous chloroform exposure and 6-week stop.
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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Animal health: No clinical signs of toxicity were noted at any time throughout the exposure study in females, and few of the male mice had rough hair coats and slight hair loss at the latter time points. No statistically significant differences between control and exposure groups were observed in body weight gain of females or males exposed for 13 weeks.
Organ weights: Relative liver weights were increased at each necropsy time point in female and male mice exposed to 90 ppm. No differences in relative kidney weights were observed between control and exposure groups at any time point.
Liver and kidney histopathology and cell proliferation: After 13 weeks of 7 days/week exposure, microscopic liver alterations (mild degenerative changes of slight centrilobular hepatocyte swelling and vacuolation) were present in 4/14 female mice exposed to 10 ppm and 10/15 mice exposed to 30 ppm. All female mice exposed to 90 ppm chloroform for 13 weeks at 5 days/week had mild changes of hepatocyte vacuolation and scattered enlarged nuclei. Increased cell proliferation was not observed at any time point with doses of 10 ppm or lower, but occurred at 3 and 6 weeks with doses of 30 ppm and at 13 weeks with doses of 90 ppm. Lesion scores for male mice exposed for 13 weeks at 7 days/week were increased above background at 30 and 90 ppm. Swollen centrilobular hepatocytes with pale eosinophilic cytoplasm, enlarged nuclei and centrilobular to midzonal hepatocyte vacuolation were observed. Significant increases in hepatocyte labelling index were found only in male mice exposed to 90 ppm.
Kidneys from female mice exposed to chloroform were not different histologically from controls at any time point. There was no statistically significant increase in the labelling index over controls in any of the treatment groups in the kidneys. Kidney lesions in male mice were confined to the epithelial cells of the proximal convoluted tubules of the cortex. Male mice exposed to 30 or 90 ppm had single or multiple foci of regeneration within the cortex. Mineralisation within the cortex was present in male mice exposed to 90 ppm. Enlarged nuclei were noted in PCT epithelial cells in male mice exposed to 10, 30 and 90 ppm. An increase in the labelling index was found in the epithelial cells of the PCT in the cortex and in the outer stripe of the outer medulla with 30 and 90 ppm, but not with 10 ppm or lower.
Nasal histopathology and cell proliferation: Nasal tissue had mild changes confined to the posterior ventral areas of the nose in and adjacent to the sites of attachment of the ethmoid turbinates to the lateral wall with doses of 10, 30 or 90 ppm. Nasal lesions were characterised by mild proliferative responses in the periosteum of the frontal and maxillary bones adjacent to the attachment of the osseous cores of the ethmoid turbinates. The response was characterised by a thickening of the normally delicate and regularly structured bone in this site through hypertrophy and hyperplasia of the periosteum and irregular formation of new, immature bone. The adjacent lamina propria exhibited variable loss of acini of Bowman's glands with mild vascular congestion and oedema.
Effects on other tissues: No microscopic changes were seen in the other tissues examined.

Key result
Dose descriptor:
NOAEC
Effect level:
9.8 mg/m³ air
Sex:
male
Basis for effect level:
other: based on renal and nasal changes at the next higher concentration of 49 mg/m3
Key result
Critical effects observed:
not specified

Table 3: Relative liver and kidney weight of female and male B6C3F1 mice exposed to chloroform vapours

13 weeks of exposure

Concentration (ppm)

Liver

Kidney

Female mice

0

5.3 ± 0.3 b)

1.7 ± 0.1 b)

0.3

5.1 ± 0.3 b)

1.6 ± 0.2 c)

2

5.0 ± 0.2 b)

1.5 ± 0.1 b)

10

5.1 ± 0.2 b)

1.5 ± 0.2 b)

30

5.6 ± 0.7 b)

1.7 ± 0.1 b)

90

6.7 ± 0.3 b)*

1.6 ± 0.2 b)

Male mice

0

4.9 ± 0.3 b)

2.2 ± 0.1 b)

0.3

4.9 ± 0.3 b)

2.1 ± 0.2 b)

2

5.1 ± 0.3 b)

2.2 ± 0.1 b)

10

5.3 ± 0.2 b)

2.4 ± 0.2 b)

30

5.5 ± 0.1 b)*

1.9 ± 0.1 b)

90

6.3 ± 0.8 b)*

2.2 ± 0.2 b)

a) values are presented as organ weights as % of body weight; means +/- SD; b) n=5; * statistically different from controls (Williams test, p 0.05) Table 4: Hepatic lesion scores and incidence in female and male maic exposed to chloroform vapours a)

Female mice

Male mice

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

0

0.1 (1/15, 1.0)

0.1 (1/15, 1.0) b)

0.1 (1/15, 1.0) b)

0.3 (5/15, 1.0)

0.3 (5/15, 1.0) b)

0.3 (5/15, 1.0) b)

0.3

0.3 (1/15, 1.0)

c)

c)

0.4 (4/15, 1.5)

c)

c)

2

0.5 (5/14, 1.4)

c)

c)

0.3 (5/14, 1.0)

c)

c)

10

0.6 (4/14, 2.0)

0.2 (2/13, 1.0)

c)

0.3 (5/15, 1.0)

0.5 (4/13, 1.5)

c)

30

0.8 (10/15, 1.2)

c)

0.3 (1/8, 2.0)

1.2 (12/12, 1.2)

c)

0.6 (4/8, 1.3)

90

2.9 (15/15, 2.9)

1.8 (13/13, 1.8)

0.8 (2/8, 3.0)

1.9 (14/14, 1.9)

0.8 (10/12, 1.3)

0.8 (4/8, 1.5)

a) chloroform-induced liver histopathological changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of the lesions ranging from lipid vacuolation to degenerative changes and necrosis. The top number is the mean lesion score for the entire group. The ratio in parentheses is that of the number of animals present with lesion relative to the total number of animals evaluated in that group. The second number in the parentheses is the mean lesion score for only that subset of animals with liver lesions; b) control animals are the same for all the 13 -week studies; c) animals were not examined at these time points. Table 5) Hepatocyte labelling indices in female and male mice exposed to chloroform vapours

Female mice

Male mice

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

0

1.3 ± 1.0 e)

1.3 ± 1.0 g)

1.3 ± 1.0 g)

0.5 ± 0.4 e)

0.5 ± 0.4 g)

0.5 ± 0.4 g)

0.3

1.4 ± 1.0 e)

h)

h)

1.1 ± 0.8 e)

h)

h)

2

0.8 ± 0.3 f)

h)

h)

0.8 ± 0.6 f)

h)

h)

10

2.4 ± 1.5 f)

0.8 ± 0.3 c)

h)

1.0 ± 0.6 f)

1.5 ± 0.9 c)

h)

30

1.8 ± 1.4 f)

h)

1.0 ± 0.6 d)

0.9 ± 0.6 f)

h)

0.9 ± 0.7 d)

90

18.1 ± 8.5 e)*

7.1 ± 3.7 c)*

0.8 ± 0.6 c)

10.7 ± 4.9 e)*

3.1 ± 2.4 c)*

1.4 ± 0.9 c)

c) n=8; d) n=7; e) n=9; f) n=10; g) control animals are the same for all the 13 -week studies; h) animals were not examined at these time points; * statistically different from control (Williams test, p 0.05) Table 6) Severity of nasal lesions in female mice exposed to chloroform vapours a)

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

0

0.1 ± 0.3 e)

0.1 ± 0.3 g)

0.1 ± 0.3 g)

0.3

0 e)

h)

h)

2

0.1 ± 0.3 e)

h)

h)

10

0.4 ± 0.5 f)

0.4 ± 0.5 d)

h)

30

0.4 ± 0.5 e)

h)

0 d)

90

0.1 ± 0.3 c)

0.3 ± 0.5 d)

0 d)

a) chloroform-induced nasal histologic changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of connective tissue proliferation in the lamina propria. The severity of the nasal lesion for each group is expressed as the mean score +/- SD; c) n=8; d) n=7; e) n=9; f) n=10; g) control animals are the same for all the 13 -week studies; h) animals were not examined at these time points Table 7: Nasal turbinate lamina propria labelling indices in female mice exposed to chloroform vapours a)

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

0

27 ± 9 e)

27 ± 9 g)

27 ± 9 g)

0.3

29 ± 27 e)

h)

h)

2

29 ± 17 d)

h)

h)

10

67 ± 33 d)

27 ± 18 b)

h)

30

43 ± 29 e)

h)

18 ± 8 b)

90

27 ± 6 b)

35 ± 8 e)

14 ± 5 e)***

a) all values are expressed as means +/- SD, b) n=5; d) n=7; e) n=4; g) control animals are the same for all the 13 -week duration studies; h) animals were not examined at these time points; *** statistically significant decreased compared to controls (Williams test, p 0.05) Table 8: Renal lesion scores and incidence in male mice exposed to chloroform vapours a)

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

0

0.0 (0/15, 0.0)

0.0 (0/15, 0.0) b)

0.0 (0/15, 0.0)

0.3

0.0 (0/15, 0.0)

c)

c)

2

0.0 (0/14, 0.0)

c)

c)

10

0.0 (0/15, 0.0)

0.1 (1/13, 1.0)

c)

30

0.9 (11/12, 1.0)

c)

1.0 (8/8, 1.0)

90

1.5 (14/14, 1.5)

1.6 (12/12, 1.6)

1.3 (8/8, 1.3)

a) chloroform-induced kidney histologic changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of nephropaty, which includes degenerative changes, foci of regeneration and mineralisation within the cortex. The top number is the mean lesion score for the entire group. The ratio in parentheses is that of the number of animals present with a kidney lesion relative to the total number of animals evaluated in that group. The second number in the parentheses is the mean lesion score for only that subset of animals with kidney lesions; b) control anaimals are the same for all the 13 -week studies; c) animals were not examined at these time points Table 9: Labelling indices in sections of the kidneys in male mice exposed to chloroform vapours a)

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

Cortex

0

1.6 ± 0.6 f)

1.6 ± 0.6 g)

1.6 ± 0.6 g)

0.3

1.9 ± 0.8 f)

h)

h)

2

1.6 ± 0.6 e)

h)

h)

10

2.0 ± 0.9 f)

7.6 ± 3.0 c)*

h)

30

2.4 ± 1.2 f)*

h)

1.3 ± 0.4 c)

90

3.0 ± 0.9 e)*

5.7 ± 2.2 c)*

1.8 ± 0.3 c)

Outer stripe of outer medulla

0

0.8 ± 0.3 f)

0.8 ± 0.3 g)

0.8 ± 0.3 g)

0.3

1.1 ± 0.3 f)

h)

h)

2

0.8 ± 0.3 e)

h)

h)

10

0.8 ± 0.3 f)

1.6 ± 0.8 c)*

h)

30

0.6 ± 0.3 f)

h)

1.0 ± 0.5 c)

90

1.0 ± 0.4 e)

1.4 ± 0.6 c)*

0.9 ± 0.2 c)

a) values are expressed as means +/- SD; c) n=8; e) n=9; f) n=10; g) control animals are the same for all the 13 -week studies; h) animals were not examined at these time points; * statistically different from control (Williams test, p 0.05)

Conclusions:
Inhalation exposure of B6C3F1 mice to chloroform caused mild nasal lesions of the ethmoid region, liver cell proliferation and kidneys cell proliferation in female and male mice; the NOAEC was 2 ppm (9.8 mg/m3).
Executive summary:

A 90-day repeated dose inhalation toxicity study was carried out with chloroform using male and female B6C3F1 mice exposed to chloroform vapours at concentrations of 0, 0.3, 2, 10, 30 or 90 ppm (0, 1.47, 9.8, 49, 147 and 441 mg/m3) for 6 hours/day for 5 or 7 days per week. The liver of male and female animals was affected by exposure in a dose-dependent manner. The kidney of male mice was also affected by exposure. The exposure setting, i.e. groups having regular exposure interruptions for two days/week and groups receiving continuous exposure for 7 days a week and the duration of exposure, had an influence on the observed systemic effects. Increases in the relative liver weight in male and female mice were seen only at exposure to 90 ppm (441 mg/m3) chloroform. Liver lesions in male and female mice occurred at exposure concentrations of 30 (147 mg/m3) ppm or greater. The NOEC for regenerative cell proliferation in the liver of female mice was 10 ppm (49 mg/m3) and in the liver of male mice was 90 ppm (441 mg/m3). Kidneys of female mice were not different histologically from controls at any exposure concentration or time point. In contrast, lesions in the kidneys of male mice (enlarged nuclei in PCT epithelial cells and scattered areas of regenerating foci) were observed at exposure concentrations of 10 ppm (49 mg/m3). At 13 weeks, increased regenerative cell proliferation in the kidneys occurred at 30 ppm (147 mg/m3) when male mice were exposed continuously for 7 days a week and at 10 ppm (49 mg/m3) when male mice were exposed for 5 days a week. Local effects in the respiratory tract consisting of nasal lesions and induced cell proliferation essentially were confined to male and female mice exposed to concentrations of 10 ppm (49 mg/m3) or greater. These effects were observed only at the early time point after 4 days and were transient during the remainder of the study. In conclusion, male mice tended to be more susceptible to inhalation exposure to chloroform vapours than female mice. Male mice exhibited lesions of the kidney cells and increased regenerative cell proliferation in the kidneys at exposure concentration of 10 ppm (49 mg/m3), which was the LOAEC in the study; the NOAEC was set at 2 ppm (9.8 mg/m3).

Endpoint:
sub-chronic toxicity: inhalation
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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
7-week old male and female F-344 rats (CDF(F-344)/CrBR) were obtained from Charles River Breeding Laboratories, Inc., Raleigh, North Carolina, USA; rats were housed one per cage in 8 m3 stainless steel and glass inhalation chambers; animals were randomised by weight and assigned to control or treatment groups with separate chambers used for each exposure concentration; chambers were maintained on a 12-hr light-dark cycle; temperature was at 22.2 +/- 2 °C, humidity was 50 +/- 10 %, chambers were operated with a continuous flow of HEPA- and charcoal filtered air at 2000 litre/minute; rats were acclimated in the chambers for 2 weeks and were 9-weeks old at the start of exposure; NIH-07 rodent chow (Ziegler Bros., Gardener, Pennsylvania, USA) and deionised filtered tap water were available ad libitum; food was changed daily; animals were weighed before the beginning of the test and then biweekly
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
Exposure atmosphere was generated by a vaporisation technique; Nitrogen, metered by a mass flow controller, was admitted into the chloroform storage vessel through a dip tube; the chloroform-containing nitrogen gas flowed into the supply air duct for the exposure chamber; in the 2 and 10 ppm chambers, nitrogen was used to pressurise the vessel to 5 psi and carry the chloroform into the chamber through a mass flow controller
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of chloroform were monitored using a Miran 1A infrared gas analyser; average analytical exposure concentrations were always within 4.5 % of the target with standard deviations of no more than 2.7 % from the mean
Duration of treatment / exposure:
Daily exposures were conducted for 6 hours, for duration of treatment see Table 1
Frequency of treatment:
see Table 1
Dose / conc.:
9.8 mg/m³ air
Remarks:
2 ppm (target concentration)
Dose / conc.:
49 mg/m³ air
Remarks:
10 ppm (target concentration)
Dose / conc.:
147 mg/m³ air
Remarks:
30 ppm (target concentration)
Dose / conc.:
441 mg/m³ air
Remarks:
90 ppm (target concentration)
Dose / conc.:
1 470 mg/m³ air
Remarks:
300 ppm (target concentration)
No. of animals per sex per dose:
see Table 1
Control animals:
yes
Details on study design:
see Table 1
Sacrifice and pathology:
Rats in unlabelled group were weighed and killed subsequently; livers and kidneys were removed, weighed and examined macroscopically, then slides were prepared from the tissues; in rats exposed for 3 or 13 weeks, a complete tissue screen was collected including adrenals, brain, cecum, cervix, colon, duodenum, ear canal, esophagus, eye with haderian gland, femoral-tibial joint, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mesenteric lymph nodes, ovaries, pancreas, parathyroid gland, ribs, prostate, salivary gland, skin with mammary gland, sciatic nerve, seminal vesicles, spinal cord, sternum, stomach, spleen, testes, thigh muscle, thymus, thyroid, trachea, urinary bladder, uterus, vagina and vertebrae.
Other examinations:
Nasal cavities were examined as well
Statistics:
The Williams test was used to determine significant differences in organ weights, bodyweights and labelling index between treatment and control groups; the student's t-test was used to determine statistical differences in bodyweight and organ weights and labelling index between the exposure groups of 7 and 5 days/week and between exposure groups of 13-week continuous chloroform exposure and 6-week stop.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Signs of mild dehydration were observed in some rats at early time points, slight hair loss, discharge from the eyes and anogenital staining were noted at later time points. These observations were confined primarily to the high dose groups. Dose-dependent decreases in body weight gain were observed in chloroform-exposed rats at all time points. This effect was most pronounced in animals exposed to 90 or 300 ppm. Significant increases in kidney and liver weights were observed in animals exposed to 90 or 300 ppm, with effects more pronounced in female rats. Scattered vacuolation of the PCT and enlarged epithelial cell nuclei were observed in male rats exposed to 90 ppm (441 mg/m3). Necrosis of individual tubule cells was found in male rats exposed to 300 ppm (1470 mg/m3). Increased regenerative cell proliferation in the kidneys of male rats was seen with exposure concentrations of 30 ppm (147 mg/m3) or greater. In female rats significant lesions in the kidneys were observed at 300 ppm (1470 mg/m3), however, histologic changes such as vacuolation in the cortex were seen at exposure levels of 30 ppm (147 mg/m3) and greater. As in the male rats, increased regenerative cell proliferation was observed in the kidneys of female rats at exposure to 30 ppm (147 mg/m3) or greater. Thus, the NOAEC for adverse effects in the kidneys was 10 ppm (49 mg/m3) for both male and female rats. Hepatocyte alterations were found in male and female rats exposed to 90 (441 m/m3) or 300 ppm (1470 mg/m3). An increase in regenerative cell proliferation in the liver of male and female rats was observed only at exposure to 300 ppm (1470 mg/m3). In conclusion, repeated inhalation exposure to chloroform vapours for 13 weeks resulted in local and systemic effects in male and female F-344 rats.
Key result
Dose descriptor:
NOAEC
Effect level:
49 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: based on renal changes at the next higher level of 147 mg/m3
Key result
Critical effects observed:
not specified
Organ:
kidney

Table 2: Kidney lesion scores and incidence in male and female F-344 rats exposed to chloroform vapours

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

Male rats

0

0.6 (8/14)

0.6 (8/14) b)

0.6 (8/14) b)

2

0.8 (10/15)

c)

c)

10

0.5 (7/15)

c)

c)

30

0.6 (9/14)

0.1 (2/15)

c)

90

1.2 (14/15)

0.6 (6/13)

1.1 (8/8)

300

1.4 (14/14)

2.8 (13/13)

1.4 (8/8)

Female rats

0

0.4 (6/14)

0.4 (6/14) b)

0.4 (6/14) b)

2

0.7 (10/15)

c)

c)

10

0.7 (10/15)

c)

c)

30

0.8 (12/15)

1.8 (13/13)

c)

90

0.7 (10/15)

0.4 (5/13)

0.9 (7/8)

300

1.1 (14/14)

1.4 (13/13)

0.8 (6/8)

a) chloroform-induced kidney histopathological changes were scored qualitatively as follows: 0 = within normal limits; 1 0 minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of the lesions ranging from vacuolation of proximal cell tubule (PCT) epithelium, enlarged PCT nuclei, pyknotic PCT nuclei, to individual tubule cell necrosis. The first number in each box is the mean lesion score for the entire group. The ratio in parentheses is that of the number of animals present with a lesion score of 1 or greater relative to the total number of animals evaluated in the group; b) control animals are the same for all the 13 -week studies; c) animals were not exposed at these time points Table 3: Hepatic lesion scores and incidence in male and female F-344 rats exposed to chloroform vapours

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

Male rats

0

0.1 (1/15)

0.1 (1/15) b)

0.1 (1/15) b)

2

0.2 (3/15)

c)

c)

10

0.0 (0/15)

c)

c)

30

0.1 (2/15)

0.0 (0/13)

c)

90

1.0 (14/15)

0.3 (4/13)

0.0 (0/8)

300

3.9 (15/15)

2.4 (13/13)

0.0 (0/8)

Female rats

0

0.1 (1/15)

0.1 (1/15) b)

0.1 (1/15) b)

2

0.1 (1/15)

c)

c)

10

0.0 (0/14)

c)

c)

30

0.0 (0/15)

0.0 (0/13)

c)

90

0.8 (12/15)

0.3 (4/13)

0.1 (1/8)

300

3.0 (15/15)

2.0 (13/13)

0.0 (0/8)

a) chloroform-induced liver histopathological changes were scored qualitatively as follows: 0 = within normal limits; 1 0 minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of the lesions ranging from hepatocyte vacuolation, degenerative changes in hepatocytes to hepatocyte necrosis. The first number in each box is the mean lesion score for the entire group. The ratio in the parentheses is that of the number of animals present with a lesion score of 1 or greater relative to the total number of animals evaluated in the group; b) control animals are the same for all the 13 -week studies; c) animals were not exposed at these time points Table 4: Severity of nasal lesions in male F-344 rats exposed to chloroform vapours (effects in female rats were similar but not reported in the original publication).

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

0

0.0 (0/10)

0.0 (0/10) b)

0.0 (0/10) b)

2

1.1 (10/10)

c)

c)

10

2.0 (10/10)

c)

c)

30

2.0 (10/10)

1.8 (8/8)

c)

90

2.5 (10/10)

2.0 (8/8)

2.1 (5/8)

300

2.9 (10/10)

3.0 (8/8)

2.9 (8/8)

a) chloroform-induced histopathological changes in the ethmoid region of the nasal passage were scored qualitatively as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of the lesions. Severity scores were assigned for lesions ranging from oedemal and loss of Bowman's glands, olfactory metaplasia, basal lamina mineralisation to generalised atrophy of the ethmoid turbinates. The first number in each box is the mean lesion score for the entire group. The ratio in parentheses is that of the number of animals present with a lesion score of 1 or greater relative to the total number of animals evaluated in the group; b) control animals are the same for all the 13 -week studies; c) animals were not exposed at these time points

Conclusions:
Based on the results of this study, the NOAEC was set at 10 ppm (49 mg/m3) based on renal changes in male and female rats.
Executive summary:

A 90-day subchronic toxicity study was carried out in male and female F-344 rats according to principles similar to those of the OECD Guideline for the Testing of Chemicals No. 413 (with acceptable restrictions). Different groups of rats were exposed by inhalation to chloroform vapours at concentrations of 0, 2, 10, 30, 90, 300 ppm (0, 9.8, 49, 147, 441, 1470 mg/m3) for 6 hours per day for 5 or 7 days per week for 13 weeks. Systemic effects were seen in the kidneys and the liver of rats. At 13 weeks of exposure, scattered vacuolation of the PCT and enlarged epithelial cell nuclei were observed in male rats exposed to 90 ppm (441 mg/m3). Necrosis of individual tubule cells was found in male rats exposed to 300 ppm (1470 mg/m3). Increased regenerative cell proliferation in the kidneys of male rats was seen with exposure concentrations of 30 ppm (147 mg/m3) or greater. In female rats exposed for 13 weeks, significant lesions in the kidneys were observed at 300 ppm (1470 mg/m3) levels, however, histologic changes such as vacuolation in the cortex were seen at exposure levels of 30 ppm (147 mg/m3) and greater. As in the male rats, increased regenerative cell proliferation was observed in the kidneys of female rats at exposure to 30 ppm (147 mg/m3) or greater. Thus, the NOAEC for adverse effects in the kidneys was 10 ppm (49 mg/m3) for male and female rats, respectively. At 13 weeks of exposure, hepatocyte alterations were found in male and female rats exposed to 90 (441 mg/m3) or 300 ppm (1470 mg/m3). An increase in regenerative cell proliferation in the liver of male and female rats was observed only at exposure to 300 ppm (1470 mg/m3). In conclusion, repeated inhalation exposure to chloroform vapours for 13 weeks resulted in local and systemic effects in male and female F-344 rats. The NOEC for increased regenerative cell proliferation and lesions in the kidneys of male and female rats was 10 ppm (49 mg/m3).

Endpoint:
sub-chronic toxicity: inhalation
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
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
other: BDF1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Raleigh
- Age at study initiation: 9 weeks
- Weight at study initiation: between 24.9 and 25.9 g (means for 8 mice per exposure group)
- Fasting period before study: not given
- Housing: one per cage
- Diet (e.g. ad libitum): NIH-07 rodent chow (Ziegler Bros., Gardener Bros., Pennsylvania), ad libitum
- Water (e.g. ad libitum): deionised, filtered tap water ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 50 +/- 10
- Air changes (per hr): 13.5
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
Exposures were conducted in 1-m3 chambers (H1000, Lab Products, Inc., Maywood) contained within 8-m3 chambers. A continuous flow of HEPA-filtered air was pulled through the chambers. The exposure atmosphere was generated by a vaporisation technique. Chloroform was stored in stainless steel pressure vessels. Nitrogen was used to pressurise the vessel to 5-8 p.s.i. and carry the chloroform vapours through a mass flow controller into the chamber air inlet.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations were monitored by gas chromatography (Model 5890, Hewlett-Packard Co.). The chromatograph was calibrated using certified gas standards that spanned the target concentrations. Daily average analytical exposure concentrations were within 0.5% of the target for the 5, 30 and 90 ppm target concentrations, with a coefficient of variation of <3%. For the 1 ppm chamber, the average analytical exposure concentration was within 8% of target with a coefficient of variation of 21%. For the 10 ppm chamber, the analytical concentration was within 2% of target with a coefficient of variation of 12%, for the 10 days that the male mice were exposed.
Duration of treatment / exposure:
Groups were exposed for 3 weeks, 7 weeks, or 13 weeks
Frequency of treatment:
6 hours per day for 5 days a week
Dose / conc.:
5 mg/m³ air
Remarks:
1 ppm (target concentration)
Dose / conc.:
25 mg/m³ air
Remarks:
5 ppm (target concentration)
Dose / conc.:
147 mg/m³ air
Remarks:
30 ppm (target concentration)
Dose / conc.:
441 mg/m³ air
Remarks:
90 ppm (target concentration)
No. of animals per sex per dose:
8
Control animals:
yes, concurrent no treatment
Details on study design:
A gradual step up procedure was chosen for the 30 and 90 ppm male concentration groups. Male mice in the 30 ppm group were exposed to 5 ppm for 2 weeks, to 10 ppm for 2 weeks and to 30 ppm for the remainder of the study. Male mice in the 90 ppm dose group were exposed to 5 ppm for 2 weeks, to 10 ppm for 2 weeks, to 30 ppm for 2 weeks and to 90 ppm for the remainder of the study. No step up procedure was required for the female mice. At 3.5 days prior to the scheduled necropsy, osmotic pumps were implanted surgically to administer bromodeoxyuridine (BrdU). The osmotic pumps contained a solution of 20 mg/mL filtered-sterilised BrdU in phosphate buffered saline and were implanted under aseptic conditions.
Positive control:
No
Observations and examinations performed and frequency:
The study focussed on liver and renal histopathology and cell proliferation based on a series of preceding investigations.
Sacrifice and pathology:
At necropsy, mice were weighed. Whole liver and both kidneys were immediately removed, weighed and examined macroscopically after euthanasation by exsanguination. Longitudinal sections of the left and median lobes of the liver, a cross-section of the right kidney, and a longitudinal section of the left kidney were fixed in 10 % neutral buffered formalin. A section of the duodenum was also collected as a high turnover tissue to verify systemic delivery of BrdU. Chloroform-induced kidney hisologic changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal changes; 2 = mild changes, approx. 25 % of the cortex affected with regenerating tubules; 3 = moderate changes, approx. 25 % of the cortex affected with regenerating tubules; 4 = sever changes, over 75 % of the cortex affected with regenerating tubules. Chloroform-induced hepatocyte changes were not scored because of an absence of necrosis. Liver alterations were described with increasing severity as centrilobullar swelling, centrilobular vacuolation and degeneration, or centrilobular to midzonal vacuolation and degeneration.
Other examinations:
BrdU-labelled tissue was mounted on ProbeOn Plus Slides to ensure adhesion during processing by incubating for 1 hour at room temperature with an BrdU antibody (Becton-Dickinson, Mountain View). After incubation with primary antibody, the slides were incubated for 30 minutes at room temperature with biotinylated horse anti-mouse IgG. Slides were then incubated with an avidin-biotin peroxidase complex (Vectastain ABC peroxidase kit, Burlingame) for 30 minutes at room temperature. The BrdU incorporation was visualised by a final incubation with the chomage 3-amino-9-ethylcarbazole (Zymed, San Francisco) and counterstained with hematoxylin. The labelling index was calculated by dividing the number of nuclei that stained positive for BrdU incorporation by the total number of nuclei counted, with the result expressed as percentage. The Cytology/Histology Recognition Information System software package developed by Sverdrup Technology, Inc., was used to assess regenerative cell proliferation. In the kidney, 15 fields were collected in the cortex and outer stripe of the outer medulla. Ten fields were collected in the liver.
Statistics:
The Williams test was used to determine significant differences in organ weights, body weights and the labelling index between treatment and control groups. The Williams test is designed to establish the lowest dose that is significantly elevated over the control in a dose-response study.
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):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Female mice did not exhibit renal lesions or cell proliferation. Necrosis was not a feature of chloroform hepatotoxicity in either sex of the BDF1 mice, which is in contrast to the findings in 90-day inhalation studies with B6C3F1 mice (Larson et al. 1996). In the male mice, histopathological changes in the liver were not observed at exposure concentrations of 1 or 5 ppm. No increases in the labelling indices in the liver of male mice were observed at concentrations of 30 ppm or below. In female mice, no histopathological changes in the liver were observed at 5 ppm or below. No increases in the labelling indices were observed in female mice exposed to 30 ppm or below.
Key result
Dose descriptor:
NOEC
Effect level:
25 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No observation of histopathological changes and induced cell proliferation in the kidney
Key result
Critical effects observed:
not specified

Table 1: Changes in body weight with chlorform exposure

Chloroform concentration (ppm)

Change in body weight (%) a)

3 weeks

7 weeks

13 weeks

Male BDF1 mice

0

107.6± 3.6

117.4± 3.6

136.1± 13.4

1

102.6± 3.6

110.8± 3.1

126.4± 8.7

5

101.2± 4.7

110.8± 3.0

118.9± 4.2

30

 

110.6± 5.3

113.4± 5.7

90

 

109.8± 5.3

116.5± 6.4

Female BDF1 mice

0

114.4± 13.3

 

133.5± 12.1

5

117.6± 3.1

 

130.8± 4.1

30

115.4± 3.6

 

129.5± 6.1

90

108.2± 1.3

 

134.3± 5.7

a) Values shown are the means +/- SD for each group. Percent change is relative to initial body weight at the beginning of the study.

Table 2: Changes in liver weight with chloroform exposure

Chloroform concentration (ppm)

Liver weight as percent of body weight a)

3 weeks

7 weeks

13 weeks

Male BDF1 mice

0

5.39± 0.13

4.78± 0.35

4.32± 0.4

1

4.64± 0.84

4.62± 0.26

4.45± 0.2

5

4.87± 0.41

4.59± 0.25

4.57± 0.1

30

 

5.54± 0.24 b)

5.28± 0.4 b)

90

 

5.66± 0.23 b)

5.72± 0.3 b)

Female BDF1 mice

0

5.47± 0.17

 

4.98± 0.4

5

5.32± 0.38

 

4.94± 0.1

30

5.62± 0.10

 

5.21± 0.2

90

6.25± 0.44 b)

 

6.16± 0.3 b)

a) Values shown are the mean +/- SD for each group.

b) Significantly different from control, Williams test, p < 0.05

Table 3: Histopathological changes and scores in the kidneys of male mice exposed to chloroform

Chloroform concentration (ppm)

Histopathological scores

3 weeks

7 weeks

13 weeks

Male BDF1 mice

0

0

0.2

0

1

0.25

0.2

0.25

5

0

0.2

0.25

30

-

3

2.75

90

-

3.4

2.75
Conclusions:
The NOEC for regenerative cell proliferation in BDF1 male mouse kidney was 5 ppm (25 mg/m3) in the 90-day repeated inhalation dose study. Female mice were less susceptible with regard to chloroform exposure than male mice.
Executive summary:

A 90-day repeated dose inhalation study was performed with male and female BDF1 mice according to procedures similar to those of the EU Method B.29 with restrictions. Mice were exposed for 6 hours per day, for 5 days per week to vapour concentrations of chloroform of 0, 1, 5, 30, or 90 ppm (0, 5, 25, 147, 441 mg/m3). The study focussed on histopathological changes in the kidneys and the livers of the test animals as these endpoints were the most sensitive toxicological endpoints identified in preceding studies. Male mice were more susceptible with regard to chloroform exposure showing histopathologic changes in the kidneys at exposure concentrations of 30 ppm (147 mg/m3) and 90 ppm (441 mg/m3). The NOEC for cell proliferation and tumour formation in the male mouse kidneys was 5 ppm (25 mg/m3), which covers the most sensitive toxicological endpoints. Female mice were less susceptible with regard to chloroform exposure than male BDF1 mice. In conclusion, the NOEC established in the 90-day study on repeated inhalation exposure was 5 ppm (25 mg/m3).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
25 mg/m³
Study duration:
subchronic
Species:
mouse
Organ:
kidney
nasal cavity

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
Method according to Kano et al. (2002), Journal of Occupational Health 44, 119-124
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
other: BDF1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Inc., Kanagawa, Japan
- Age at study initiation: 6 weeks
- Housing: individually in stainless-steel wire hanging cages in stainless-steel inhalation chambers for each exposure group
- Diet (e.g. ad libitum): commercial pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) ad libitum
- Water (e.g. ad libitum): sterilised ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hours dark, 12 hours light
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
A chloroform vapour-air mixture was produced by bubbling clean air through the liquid chloroform, further diluted with clean air, and supplied to the inhalation exposure chambers
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6 hours per day on 5 days per week for 104 weeks
Frequency of treatment:
6 hours per day on 5 days per week for 104 weeks
Dose / conc.:
24.5 mg/m³ air (analytical)
Remarks:
5 ppm (analytical)
Dose / conc.:
147 mg/m³ air (analytical)
Remarks:
30 ppm (analytical)
Dose / conc.:
441.5 mg/m³ air (analytical)
Remarks:
90.1 ppm (analytical)
No. of animals per sex per dose:
50 females and 50 males
Control animals:
yes, concurrent no treatment
Details on results:
Body weight of exposed males decreased significantly, whereas the decrease remained significant in female rats only in the 90 ppm group. The incidence of renal nodules significantly increased in the 30 and 90 ppm exposed males. The absolute kidney weight of the 90 ppm exposed male group significantly increased. The incidence of renal cell carcinoma in exposed males increased in a dose-related manner and was significant in the 30 and 90 ppm groups. The exposed males exhibited significantly increased incidences of cytoplasmic basophilia, nuclear enlargement and atypical tubule hyperplasia at 30 ppm and 90 ppm. The 90 ppm exposed females exhibited a significantly increased incidence of the cytoplasmic basophilia. The difference in the incidence of hepatocellular adenomas was not statistically significant between the exposed female groups and controls, but the combined incidence of hepatocellular adenomas and carcinomas showed a significant positive trend by Peto's test. An increased incidence of total altered cell foci was found in the 90 ppm females. Significant increases in the incidences of fatty change in the 90 ppm exposed males and females and total altered cell foci in the 90 ppm exposed females were noted. Thickening of the bone at 5 ppm and above for both sexes, atrophy and respiratory metaplasia of the olfactory epithelium in the 90 ppm males and in the 5 ppm and greater exposed females were noted. Exposed males showed increased serum levels of ALP at 5 ppm and greater, BUN at 30 ppm and above, GOT and GPT at 90 ppm and total protein at 90 ppm. Significantly increased levels of triglyceride, BUN, GOT and GPT were found in the 90 ppm exposed females.
Key result
Dose descriptor:
NOAEC
Effect level:
24.5 mg/m³ air (analytical)
Sex:
male
Basis for effect level:
other: Absence of renal cell carcinoma
Key result
Dose descriptor:
LOAEC
Effect level:
24.5 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: Body weight gain, nasal lesions, serum enzyme levels
Key result
Critical effects observed:
not specified
Conclusions:
The NOEC for increase of renal cell carcinoma in male mice was 5 ppm (24.5 mg/m3). The LOAEC in both sexes for body weight gain, nasal lesions and changes in serum enzyme levels in the study was 5 ppm (24.5 mg/m3).
Executive summary:

A combined carcinogenicity/chronic toxicity study was carried out with female and male BDF1 mice being exposed to chloroform vapours of 5, 30 and 90.1 ppm (24.5, 147 and 411.5 mg/m3) for 6 hours per day on 5 days per week for 104 weeks. The body weight gain of males and females was suppressed by the exposure to 5 ppm and greater. Renal changes and lesions and an increased incidence of renal cell carcinoma in males were seen at 30 ppm and 90 ppm chloroform. The difference in the incidence of hepatocellular adenomas was not statistically significant between the exposed female groups and controls, but the combined incidence of hepatocellular adenomas and carcinomas showed a significant positive trend by Peto's test. An increased incidence of total altered cell foci was found in the 90 ppm females. Significant increases in the incidences of fatty change in the 90 ppm exposed males and females and total altered cell foci in the 90 ppm exposed females were noted. Nasal changes and changes in the serum enzyme levels were observed at 5 ppm and greater.

The NOEC for increase of renal cell carcinoma in male mice was 5 ppm (24.5 mg/m3). The LOAEC in both sexes for body weight gain, nasal lesions and changes in serum enzyme levels in the study was 5 ppm (24.5 mg/m3).

Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
Method according to Kano et al. (2002), Journal of Occupational Health 44, 119-124
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Inc., Kanagawa, Japan
- Age at study initiation: 6 weeks
- Housing: individually in stainless-steel wire hanging cages in stainless-steel inhalation chambers for each exposure group
- Diet (e.g. ad libitum): commercial pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) ad libitum
- Water (e.g. ad libitum): sterilised ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hours dark, 12 hours light
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
A chloroform vapour-air mixture was produced by bubbling clean air through the liquid chloroform, further diluted with clean air, and supplied to the inhalation exposure chambers
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6 hours per day on 5 days per week for 104 weeks
Frequency of treatment:
6 hours per day on 5 days per week for 104 weeks
Dose / conc.:
49.5 mg/m³ air (analytical)
Remarks:
10.1 ppm (analytical)
Dose / conc.:
147 mg/m³ air (analytical)
Remarks:
30 ppm (analytical)
Dose / conc.:
440.5 mg/m³ air (analytical)
Remarks:
89.9 ppm (analytical)
No. of animals per sex per dose:
50 females and 50 males
Control animals:
yes, concurrent vehicle
Details on results:
The growth rates of the 90 ppm groups of both sexes were significantly suppressed over the controls. Absolute and relative kidney weights of the female 90 ppm group were statistically significant increased. Dose-related increases in the occurrence of nuclear enlargement of the proximal tubule and dilatation of the tubular lumen in the kidney were seen in the 30 ppm and 90-ppm-exposed groups of both sexes. A significantly increased incidence of vacuolated cell foci in the 90 ppm exposed females was found. The nasal lesions included thickening of the bone and atrophy and respiratory metaplasia of the olfactory epithelium at 10 ppm and greater. Exposed males exhibited significantly decreased serum levels of triglyceride, phospholipids and creatinine at 30 ppm and greater, and total cholesterol at 90 ppm. Serum levels of BUN significantly decreased in the three exposed groups of males. GOT, GPT and gamma-GTP significantly increased in the exposed males. The exposed females exhibited decreased triglyceride and increased gamma-GTP levles in the 90 ppm group. Positive urinary glucose was observed in the 90 ppm exposed males and in the 10 ppm and above exposed females.
Key result
Dose descriptor:
LOAEC
Effect level:
49.5 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Nasal lesions, and changes in serum chemistry and urinalysis
Key result
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, viz. nasal changes, and changes in serum chemistry and urinalysis, the LOAEC was set at 10.1 ppm (49.5 mg/m3).
Executive summary:

A combined carcinogenicity/chronic toxicity study was carried out with female and male F-344 rats receiving inhalation exposure to chloroform vapour at concentrations of 10.1, 30.0, 89.9 ppm (49.5, 147, 440.5 mg/m3) during 104 weeks. The animals exposed to 10 ppm chloroform and higher showed signs of nasal toxicity and changes in serum enzyme levels as well as urinary parameters. Changes in the kidney occurred at exposures of 30 ppm and greater. The LOAEC value for nasal changes found in the study was 10.1 ppm (49.5 mg/m3), whereas 10.1 ppm (49.5 mg/m3) was the NOAEC value for changes in the kidney.

Endpoint:
sub-chronic toxicity: inhalation
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
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Seven-week old male and female B6C3F1 mice obtained from Charles River Breeding Laboratories, Inc., Raleigh, North Carolina, USA; housed one per cage in 8-m3 stainless steel and glass inhalation chambers; separate chambers for each exposure concentration; acclimation for 2 weeks; chambers maintained on a 12-hours light/12-hours dark cycle, NIH-07 rodent chow (Ziegler Bros., Gardener; Pennsylvania), and deionised filtered tap water available ad libitum; temperature at 22.2 +/- 2 °C and 50 +/- 10 % relative humidity, continuous flow of HEPA and charcoal-filtered air at flow rates of 2000 litre/min
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
Exposure atmosphere was generated by a vaporisation technique; Nitrogen, metered by a mass flow controller, was admitted into the chloroform storage vessel through a dip tube; the chloroform-containing nitrogen gas flowed into the supply air duct for the exposure chamber; in the 2 and 10 ppm chambers, nitrogen was used to pressurise the vessel to 5 psi and carry the chloroform into the chamber through a mass flow controller
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of chloroform were monitored using a Miran 1A infrared gas analyser (see Table 1)
Duration of treatment / exposure:
see Table 2
Frequency of treatment:
6 hours on 5 days/week or 7 days/week
Dose / conc.:
1.47 mg/m³ air
Remarks:
0.3 ppm (target concentration)
Dose / conc.:
9.8 mg/m³ air
Remarks:
2 ppm (target concentration)
Dose / conc.:
49 mg/m³ air
Remarks:
10 ppm (target concentration)
Dose / conc.:
147 mg/m³ air
Remarks:
30 ppm (target concentration)
Dose / conc.:
441 mg/m³ air
Remarks:
90 ppm (target concentration)
No. of animals per sex per dose:
ranging from 10 to 60, see Table 2
Control animals:
yes
Sacrifice and pathology:
Mice in unlabelled group were weighed and killed subsequently; livers and kidneys were removed, weighed and examined macroscopically, then slides were prepared from the tissues; in rats exposed for 3 or 13 weeks, a complete tissue screen was collected including adrenals, brain, cecum, cervix, colon, duodenum, ear canal, esophagus, eye with haderian gland, femoral-tibial joint, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mesenteric lymph nodes, ovaries, pancreas, parathyroid gland, ribs, prostate, salivary gland, skin with mammary gland, sciatic nerve, seminal vesicles, spinal cord, sternum, stomach, spleen, testes, thigh muscle, thymus, thyroid, trachea, urinary bladder, uterus, vagina and vertebrae.
Other examinations:
Nasal passages were examined as well
Statistics:
The Williams test was used to determine significant differences in organ weights, body weights and labelling index between treatment and control groups; the student's t-test was used to determine statistical differences in body weight and organ weights and labelling index between the exposure groups of 7 and 5 days/week and between exposure groups of 13-week continuous chloroform exposure and 6-week stop.
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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Animal health: No clinical signs of toxicity were noted at any time throughout the exposure study in females, and few of the male mice had rough hair coats and slight hair loss at the latter time points. No statistically significant differences between control and exposure groups were observed in body weight gain of females or males exposed for 13 weeks.
Organ weights: Relative liver weights were increased at each necropsy time point in female and male mice exposed to 90 ppm. No differences in relative kidney weights were observed between control and exposure groups at any time point.
Liver and kidney histopathology and cell proliferation: After 13 weeks of 7 days/week exposure, microscopic liver alterations (mild degenerative changes of slight centrilobular hepatocyte swelling and vacuolation) were present in 4/14 female mice exposed to 10 ppm and 10/15 mice exposed to 30 ppm. All female mice exposed to 90 ppm chloroform for 13 weeks at 5 days/week had mild changes of hepatocyte vacuolation and scattered enlarged nuclei. Increased cell proliferation was not observed at any time point with doses of 10 ppm or lower, but occurred at 3 and 6 weeks with doses of 30 ppm and at 13 weeks with doses of 90 ppm. Lesion scores for male mice exposed for 13 weeks at 7 days/week were increased above background at 30 and 90 ppm. Swollen centrilobular hepatocytes with pale eosinophilic cytoplasm, enlarged nuclei and centrilobular to midzonal hepatocyte vacuolation were observed. Significant increases in hepatocyte labelling index were found only in male mice exposed to 90 ppm.
Kidneys from female mice exposed to chloroform were not different histologically from controls at any time point. There was no statistically significant increase in the labelling index over controls in any of the treatment groups in the kidneys. Kidney lesions in male mice were confined to the epithelial cells of the proximal convoluted tubules of the cortex. Male mice exposed to 30 or 90 ppm had single or multiple foci of regeneration within the cortex. Mineralisation within the cortex was present in male mice exposed to 90 ppm. Enlarged nuclei were noted in PCT epithelial cells in male mice exposed to 10, 30 and 90 ppm. An increase in the labelling index was found in the epithelial cells of the PCT in the cortex and in the outer stripe of the outer medulla with 30 and 90 ppm, but not with 10 ppm or lower.
Nasal histopathology and cell proliferation: Nasal tissue had mild changes confined to the posterior ventral areas of the nose in and adjacent to the sites of attachment of the ethmoid turbinates to the lateral wall with doses of 10, 30 or 90 ppm. Nasal lesions were characterised by mild proliferative responses in the periosteum of the frontal and maxillary bones adjacent to the attachment of the osseous cores of the ethmoid turbinates. The response was characterised by a thickening of the normally delicate and regularly structured bone in this site through hypertrophy and hyperplasia of the periosteum and irregular formation of new, immature bone. The adjacent lamina propria exhibited variable loss of acini of Bowman's glands with mild vascular congestion and oedema.
Effects on other tissues: No microscopic changes were seen in the other tissues examined.

Key result
Dose descriptor:
NOAEC
Effect level:
9.8 mg/m³ air
Sex:
male
Basis for effect level:
other: based on renal and nasal changes at the next higher concentration of 49 mg/m3
Key result
Critical effects observed:
not specified

Table 3: Relative liver and kidney weight of female and male B6C3F1 mice exposed to chloroform vapours

13 weeks of exposure

Concentration (ppm)

Liver

Kidney

Female mice

0

5.3 ± 0.3 b)

1.7 ± 0.1 b)

0.3

5.1 ± 0.3 b)

1.6 ± 0.2 c)

2

5.0 ± 0.2 b)

1.5 ± 0.1 b)

10

5.1 ± 0.2 b)

1.5 ± 0.2 b)

30

5.6 ± 0.7 b)

1.7 ± 0.1 b)

90

6.7 ± 0.3 b)*

1.6 ± 0.2 b)

Male mice

0

4.9 ± 0.3 b)

2.2 ± 0.1 b)

0.3

4.9 ± 0.3 b)

2.1 ± 0.2 b)

2

5.1 ± 0.3 b)

2.2 ± 0.1 b)

10

5.3 ± 0.2 b)

2.4 ± 0.2 b)

30

5.5 ± 0.1 b)*

1.9 ± 0.1 b)

90

6.3 ± 0.8 b)*

2.2 ± 0.2 b)

a) values are presented as organ weights as % of body weight; means +/- SD; b) n=5; * statistically different from controls (Williams test, p 0.05) Table 4: Hepatic lesion scores and incidence in female and male maic exposed to chloroform vapours a)

Female mice

Male mice

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

0

0.1 (1/15, 1.0)

0.1 (1/15, 1.0) b)

0.1 (1/15, 1.0) b)

0.3 (5/15, 1.0)

0.3 (5/15, 1.0) b)

0.3 (5/15, 1.0) b)

0.3

0.3 (1/15, 1.0)

c)

c)

0.4 (4/15, 1.5)

c)

c)

2

0.5 (5/14, 1.4)

c)

c)

0.3 (5/14, 1.0)

c)

c)

10

0.6 (4/14, 2.0)

0.2 (2/13, 1.0)

c)

0.3 (5/15, 1.0)

0.5 (4/13, 1.5)

c)

30

0.8 (10/15, 1.2)

c)

0.3 (1/8, 2.0)

1.2 (12/12, 1.2)

c)

0.6 (4/8, 1.3)

90

2.9 (15/15, 2.9)

1.8 (13/13, 1.8)

0.8 (2/8, 3.0)

1.9 (14/14, 1.9)

0.8 (10/12, 1.3)

0.8 (4/8, 1.5)

a) chloroform-induced liver histopathological changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of the lesions ranging from lipid vacuolation to degenerative changes and necrosis. The top number is the mean lesion score for the entire group. The ratio in parentheses is that of the number of animals present with lesion relative to the total number of animals evaluated in that group. The second number in the parentheses is the mean lesion score for only that subset of animals with liver lesions; b) control animals are the same for all the 13 -week studies; c) animals were not examined at these time points. Table 5) Hepatocyte labelling indices in female and male mice exposed to chloroform vapours

Female mice

Male mice

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

0

1.3 ± 1.0 e)

1.3 ± 1.0 g)

1.3 ± 1.0 g)

0.5 ± 0.4 e)

0.5 ± 0.4 g)

0.5 ± 0.4 g)

0.3

1.4 ± 1.0 e)

h)

h)

1.1 ± 0.8 e)

h)

h)

2

0.8 ± 0.3 f)

h)

h)

0.8 ± 0.6 f)

h)

h)

10

2.4 ± 1.5 f)

0.8 ± 0.3 c)

h)

1.0 ± 0.6 f)

1.5 ± 0.9 c)

h)

30

1.8 ± 1.4 f)

h)

1.0 ± 0.6 d)

0.9 ± 0.6 f)

h)

0.9 ± 0.7 d)

90

18.1 ± 8.5 e)*

7.1 ± 3.7 c)*

0.8 ± 0.6 c)

10.7 ± 4.9 e)*

3.1 ± 2.4 c)*

1.4 ± 0.9 c)

c) n=8; d) n=7; e) n=9; f) n=10; g) control animals are the same for all the 13 -week studies; h) animals were not examined at these time points; * statistically different from control (Williams test, p 0.05) Table 6) Severity of nasal lesions in female mice exposed to chloroform vapours a)

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

0

0.1 ± 0.3 e)

0.1 ± 0.3 g)

0.1 ± 0.3 g)

0.3

0 e)

h)

h)

2

0.1 ± 0.3 e)

h)

h)

10

0.4 ± 0.5 f)

0.4 ± 0.5 d)

h)

30

0.4 ± 0.5 e)

h)

0 d)

90

0.1 ± 0.3 c)

0.3 ± 0.5 d)

0 d)

a) chloroform-induced nasal histologic changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of connective tissue proliferation in the lamina propria. The severity of the nasal lesion for each group is expressed as the mean score +/- SD; c) n=8; d) n=7; e) n=9; f) n=10; g) control animals are the same for all the 13 -week studies; h) animals were not examined at these time points Table 7: Nasal turbinate lamina propria labelling indices in female mice exposed to chloroform vapours a)

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

0

27 ± 9 e)

27 ± 9 g)

27 ± 9 g)

0.3

29 ± 27 e)

h)

h)

2

29 ± 17 d)

h)

h)

10

67 ± 33 d)

27 ± 18 b)

h)

30

43 ± 29 e)

h)

18 ± 8 b)

90

27 ± 6 b)

35 ± 8 e)

14 ± 5 e)***

a) all values are expressed as means +/- SD, b) n=5; d) n=7; e) n=4; g) control animals are the same for all the 13 -week duration studies; h) animals were not examined at these time points; *** statistically significant decreased compared to controls (Williams test, p 0.05) Table 8: Renal lesion scores and incidence in male mice exposed to chloroform vapours a)

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

0

0.0 (0/15, 0.0)

0.0 (0/15, 0.0) b)

0.0 (0/15, 0.0)

0.3

0.0 (0/15, 0.0)

c)

c)

2

0.0 (0/14, 0.0)

c)

c)

10

0.0 (0/15, 0.0)

0.1 (1/13, 1.0)

c)

30

0.9 (11/12, 1.0)

c)

1.0 (8/8, 1.0)

90

1.5 (14/14, 1.5)

1.6 (12/12, 1.6)

1.3 (8/8, 1.3)

a) chloroform-induced kidney histologic changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of nephropaty, which includes degenerative changes, foci of regeneration and mineralisation within the cortex. The top number is the mean lesion score for the entire group. The ratio in parentheses is that of the number of animals present with a kidney lesion relative to the total number of animals evaluated in that group. The second number in the parentheses is the mean lesion score for only that subset of animals with kidney lesions; b) control anaimals are the same for all the 13 -week studies; c) animals were not examined at these time points Table 9: Labelling indices in sections of the kidneys in male mice exposed to chloroform vapours a)

Concentration (ppm)

13 weeks, 7 days/week

13 weeks, 5 days/week

13 weeks, exposure stop after 6 weeks

Cortex

0

1.6 ± 0.6 f)

1.6 ± 0.6 g)

1.6 ± 0.6 g)

0.3

1.9 ± 0.8 f)

h)

h)

2

1.6 ± 0.6 e)

h)

h)

10

2.0 ± 0.9 f)

7.6 ± 3.0 c)*

h)

30

2.4 ± 1.2 f)*

h)

1.3 ± 0.4 c)

90

3.0 ± 0.9 e)*

5.7 ± 2.2 c)*

1.8 ± 0.3 c)

Outer stripe of outer medulla

0

0.8 ± 0.3 f)

0.8 ± 0.3 g)

0.8 ± 0.3 g)

0.3

1.1 ± 0.3 f)

h)

h)

2

0.8 ± 0.3 e)

h)

h)

10

0.8 ± 0.3 f)

1.6 ± 0.8 c)*

h)

30

0.6 ± 0.3 f)

h)

1.0 ± 0.5 c)

90

1.0 ± 0.4 e)

1.4 ± 0.6 c)*

0.9 ± 0.2 c)

a) values are expressed as means +/- SD; c) n=8; e) n=9; f) n=10; g) control animals are the same for all the 13 -week studies; h) animals were not examined at these time points; * statistically different from control (Williams test, p 0.05)

Conclusions:
Inhalation exposure of B6C3F1 mice to chloroform caused mild nasal lesions of the ethmoid region, liver cell proliferation and kidneys cell proliferation in female and male mice; the NOAEC was 2 ppm (9.8 mg/m3).
Executive summary:

A 90-day repeated dose inhalation toxicity study was carried out with chloroform using male and female B6C3F1 mice exposed to chloroform vapours at concentrations of 0, 0.3, 2, 10, 30 or 90 ppm (0, 1.47, 9.8, 49, 147 and 441 mg/m3) for 6 hours/day for 5 or 7 days per week. The liver of male and female animals was affected by exposure in a dose-dependent manner. The kidney of male mice was also affected by exposure. The exposure setting, i.e. groups having regular exposure interruptions for two days/week and groups receiving continuous exposure for 7 days a week and the duration of exposure, had an influence on the observed systemic effects. Increases in the relative liver weight in male and female mice were seen only at exposure to 90 ppm (441 mg/m3) chloroform. Liver lesions in male and female mice occurred at exposure concentrations of 30 (147 mg/m3) ppm or greater. The NOEC for regenerative cell proliferation in the liver of female mice was 10 ppm (49 mg/m3) and in the liver of male mice was 90 ppm (441 mg/m3). Kidneys of female mice were not different histologically from controls at any exposure concentration or time point. In contrast, lesions in the kidneys of male mice (enlarged nuclei in PCT epithelial cells and scattered areas of regenerating foci) were observed at exposure concentrations of 10 ppm (49 mg/m3). At 13 weeks, increased regenerative cell proliferation in the kidneys occurred at 30 ppm (147 mg/m3) when male mice were exposed continuously for 7 days a week and at 10 ppm (49 mg/m3) when male mice were exposed for 5 days a week. Local effects in the respiratory tract consisting of nasal lesions and induced cell proliferation essentially were confined to male and female mice exposed to concentrations of 10 ppm (49 mg/m3) or greater. These effects were observed only at the early time point after 4 days and were transient during the remainder of the study. In conclusion, male mice tended to be more susceptible to inhalation exposure to chloroform vapours than female mice. Male mice exhibited lesions of the kidney cells and increased regenerative cell proliferation in the kidneys at exposure concentration of 10 ppm (49 mg/m3), which was the LOAEC in the study; the NOAEC was set at 2 ppm (9.8 mg/m3).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
10 mg/m³
Study duration:
subacute
Species:
other: mouse, rat: atrophy of the ethmoid turbinates
Quality of whole database:
In the chronic inhalation study in mice and rats (Yamamoto, 2002), the LOAEC for nasal changes was 24.5 mg/m3 in mice and 49.5 mg/m3 in rats; a NOAEC of 10 mg/m3 for nasal changes was observed in a 90-day inhalation study (Larson, 1996).

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

Oral exposure

A combined repeated dose and carcinogenicity study was carried out with chloroform orally administered to male and female Beagle dogs over a period of more than 7 years (Heywood et al. 1979). The study was performed according to principles comparable to those set out in the testing guideline for method B.33 suggested by the European Commission with minor restrictions. The chloroform was contained in toothpaste and administered in form of a gelatine capsule. Chronic effects related to chloroform exposure included a significantly increased level of alanine aminotransferase level in the high-dose group throughout the study and in the low-dose group during the later phase of the study (2 dose groups were tested) and a slightly increased incidence but higher severity of aggregations of vacuolated histiocytes forming "fatty cysts" in the liver of treated dogs compared to controls. A NOAEL value could not be established and the LOAEL value found in this study was 15 mg/kg bw/day established for female and male dogs together.

In rats and mice, the liver and kidneys were identified as the main target organs for chloroform administered via the oral route. A study on the sub-acute toxicity of chloroform was carried out using female F-344 rats exposed by oral gavage to graded doses of chloroform dissolved in corn oil for 5 days per week for 3 consecutive weeks (Larson et al. 1995a). The study was comparable to a guideline study according to method B.7 of the European Commission with acceptable restrictions. Mild degenerative centrilobular changes and a dose-dependent increase in the hepatocyte labelling index (% of cells in S-phase) and a dose-dependent increase in the labelling index in the kidneys were observed at 100 mg/kg body weight/day of chloroform after 3 weeks of exposure. The NOAEL for systemic effects was 34 mg/kg bw/day based on the finding of lesions and increased cell replication in the liver and kidneys at higher dose levels. Male F-344 rats exposed by oral gavage to chloroform in a similar manner exhibited comparable adverse effects in the liver and kidneys (Larson et al. 1995). However, male rats were less sensitive to chloroform exposure compared to female rats and the NOAEL established for male F-344 rats was 90 mg/kg bw/day. In a 90 -day study by Chu et al. (1982), an NOAEL of ca. 40 mg/kg bw/day was found.

Hepatotoxicity was also observed in a 90-day study with male and female B6C3F1 mice with an NOAEL of 60 mg/kg bw/day (Bull et al. 1986). The liver was also the primary target organ for chloroform toxicity in a sub-acute study carried out with female B6C3F1 mice being exposed to chloroform in corn oil for 5 days per week for 3 weeks (Larson et al. 1994a, Larson et al. 1994b). The NOAEL for hepatic degenerative changes indicated by liver histology and serum enzyme levels was 10 mg/kg bw/day and the NOAEL for hepatic cell proliferation was 34 mg/kg bw/day. In male B6C3F1 mice administered chloroform in corn oil by oral gavage for 5 days per week for 3 weeks, also the liver and kidneys were affected. The LOAEL for renal toxicity was 34 mg/kg bw/day, the NOAEL for hepatic toxicity and cell replication in the liver and the kidneys was 34 mg/kg bw/day (Larson et al., 1994c).

A 90 -day repeated dose oral toxicity study with chloroform dissolved in 10% Emulphor in deionised water was performed in male and female CD-1 mice. In this a NOAEL of 50 mg/kg bw/day was found based on renal and liver toxicity (Munson et al. 1982). A sub-acute repeated dose oral toxicity study in male CD-1 mice receiving chloroform in corn oil for 14 days a LOEL of 37 mg/kg bw/day was observed based on renal and liver toxicity (Condie et al. 1983).

 

Dermal exposure

Studies on dermal repeated exposure to chloroform are not available.

Inhalation exposure

A reliable combined chronic repeated dose/carcinogenicity study by the inhalation route was performed with male and female BDF1 mice (Yamamoto et al. 2002). Animals received whole-body exposure to chloroform vapours for 6 hours/day for 5 days per week over a period of 104 weeks. Animals exposed to 25 mg/m3 chloroform vapours showed reduced body weight gain, nasal lesions and changes in serum enzyme levels. The NOEC for renal cell carcinoma in male mice as the most sensitive carcinogenic endpoint was 25 mg/m3. A 90-day repeated dose inhalation study was performed with male and female BDF1 mice according to procedures similar to those of the EU Method B.29 with restrictions (Templin et al. 1998). Mice were exposed for 6 hours per day, for 5 days per week to vapour concentrations of chloroform of 0, 5, 25, 147, 441 mg/m3. The study focussed on histopathological changes in the kidneys and the livers of the test animals as these endpoints were the most sensitive toxicological endpoints identified in preceding studies. Male mice were more susceptible with regard to chloroform exposure showing histopathologic changes in the kidneys at exposure concentrations of 147 and 441 mg/m3. The NOAEC for cell proliferation in the male kidneys was 25 mg/m3, which covers the most sensitive toxicological endpoint. Female mice were less susceptible with regard to chloroform exposure than male BDF1 mice.

A 90 -day repeated dose inhalation toxicity study was carried out with chloroform using male and female B6C3F1 mice exposed to chloroform vapours at concentrations of 0, 1.5, 10, 50, 147 or 441 mg/m3 for 6 hours/day for 5 or 7 days per week (Larson et al. 1996). The liver of male and female animals was affected by exposure in a dose-dependent manner; the kidney of male mice was also affected by exposure. The NOAEC for liver lesions in male and female mice was 50 mg/m3, the NOAEC for regenerative cell proliferation in the liver of female and male mice was 50 mg/m3 and 441 mg/m3, respectively. The NOAEC for lesions in the kidneys of male mice was 10 mg/m3. At 13 weeks, increased regenerative cell proliferation in the kidneys occurred at 147 mg/m3 when male mice were exposed continuously for 7 days a week and at 50 mg/m3 when male mice were exposed for 5 days a week. Local effects in the respiratory tract consisting of nasal lesions and induced cell proliferation essentially were confined to male and female mice exposed to concentrations of 50 mg/m3 or greater. These effects were observed only at the early time point after 4 days and were transient during the remainder of the study. In conclusion, male mice tended to be more susceptible to inhalation exposure to chloroform vapours than female mice. The NOAEC for lesions in kidney cells and increased regenerative cell proliferation in the kidneys of male mice was 10 mg/m3. In another 90-day study (Kasai et al., 2002) in mice, the lowest concentration of 58.8 mg/m3 was considered an LOAEC.

A 90-day subchronic toxicity study was carried out in male and female F-344 rats according to principles similar to those of the relevant OECD guideline (Templin et al. 1996b). Different groups of rats were exposed by inhalation to chloroform vapours at concentrations of 0, 10, 50, 147, 441 or 1470 mg/m3 for 6 hours per day for 5 or 7 days per week for 13 weeks. Systemic effects were found in the kidneys and the liver of rats. The NOEC for renal toxicity and increased cell replication in the kidneys of male and female rats was 50 mg/m3. The NOAEC for liver toxicity in male and female rats was 147 mg/m3 and the NOAEC for increased liver cell replication was 441 mg/m3. Local effects in the respiratory tract of male and female rats were found consisting of atrophy in the lamina propria and the olfactory epithelium at 50 mg/m3 and higher. In another 90 -day study in rats (Kasai et al., 2002), the NOAEC for systemic effects was 122.5 mg/m3; this concentration was a LOAEC for local (nasal) effects. In the chronic study in rats by Yamamoto et al. (2002), the LOAEC was set at 49.5 mg/m3.

 

Effects in the nasal passage

Local changes in the respiratory tract induced by chloroform vapours were reported from acute, sub-acute (Larson et al. 1996), sub-chronic (Templin et al. 1996) and chronic (Yamamoto et al. 2002) inhalation studies. Effects included nasal lesions and cell proliferation in the olfactory epithelium and the nasal passage. Cell proliferation was observed in the nose (ethmoid region) of male F-344 rats following inhalation exposure at 10 mg/m3 for 6 h/day for 4 days, however, at 50 mg/m3 only minimal to mild lesions were seen and the effects were transient (Templin et al. 1996b). A late atrophy was present not only in the areas of early cell proliferation but in the whole ethmoid region of the nasal cavity after 13 weeks of exposure. The atrophy was minimal at exposure levels of 10 mg/m3 and more distinct at higher exposure concentrations. Following exposure to 50 mg/m3 for 6 h/day for 7 consecutive days, male F-344 rats had lesions in nasal turbinates, including increased cell proliferation in central, proximal, and distal regions of the first endoturbinate, and histological changes in the central turbinate bone. Cell proliferation was also seen in the nasal turbinates of female B6C3F1 mice exposed at 10 mg/m3, but not at 1.5 mg/m3, for 6 h/day for four consecutive days (Larson et al., 1996). Increased cell proliferation was detected in the first endoturbinate of the nasal passage in female B6C3F1 mice exposed to 50 mg chloroform/m3, 6 h/day, for 7 consecutive days (Méry et al., 1994), but no microscopic damage was seen in the nasal passages of female B6C3F1 mice exposed to up to 1500 mg/m3 for 6 h/day for 7 consecutive days. Thus, it appears that notable effects of chloroform on the respiratory tract occur with exposure concentrations of 50 mg/m3 and higher.

Justification for classification or non-classification

According to Commission Regulation (EU) No 944/2013 of October 2013, amending Annex VI to the Regulation (EC) No 1272/2008 (CLP), chloroform is classified as STOT Rep. Exp. 1, with the hazard statement H372: causes damage to organs (liver, kidneys, nasal passages) through prolonged or repeated exposure via the inhalation route.

Significant effects were observed in the repeated inhalation studies at levels below 200 mg/m3 (cut off value for 90 -day vapour studies for classification as STOT RE 1) thus requiring classification

as STOT Rep. Exp. 1, with the hazard statement H372: causes damage to organs (liver, kidneys, nasal passages) through prolonged or repeated exposure via the inhalation route.

However, for the oral route, at which significant effects were seen between 10 and 100 mg/kg bw/day (cut off values for 90 -day oral studies for classification as STOT RE 2), classification as STOT Rep. Exp. 2 would apply with the hazard statement H373: may cause damage to organs (liver, kidneys) through prolonged exposure via the oral route.

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