3-chloroaniline

Endpoint summary

Description of key information

The substance 3-chloroaniline  does not exhibit repeated dose toxicity by the oral,inhalation and dermal route.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Principles of method if other than guideline:

A subchronic study was conducted to evaluate the toxic effects of repeated administration of 3-chloroaniline to F344 rat by oral route.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Breeding Laboratories
- Age at study initiation:7-weeks-old
- Housing:Rats were housed five per cage by sex
- Diet (e.g. ad libitum):NIH-07 Open Formula Diet in pellet form were available ad libitum.
- Water (e.g. ad libitum):tap water,ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C):72+/-3 degF
- Humidity (%):50+/-15%
- Air changes (per hr):10 room air changes per hr.
- Photoperiod (hrs dark / hrs light):Fluorescent lights were on for 12 h/day.

Route of administration:

oral: gavage

Vehicle:

other: Hydrochloric acid

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:Doses were formulated for oral gavage in deionized water containing 0.1 N hydrochloric acid (pH ~2).The dose concentrations for this study were 0, 2, 4, 8, 16, and 32 mg/ml for rats.


DIET PREPARATION
- Rate of preparation of diet (frequency):Twice
- Mixing appropriate amounts with (Type of food):. NIH-07 feed
- Storage temperature of food:5 degC

VEHICLE
- Justification for use and choice of vehicle (if other than water):Hydrochloric acid
- Concentration in vehicle:5 ml/kg
- Amount of vehicle (if gavage):0, 2, 4, 8, 16, and 32 mg/ml for rats

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Once a day, 5 days/week, for 13 weeks

Remarks:

Doses / Concentrations:
0, 10, 20, 40, 80, or 160 mg/kg/day
Basis:

No. of animals per sex per dose:

0 mg/kg/day - 10 male and 10 female rats
10 mg/kg/day - 10 male and 10 female rats
20 mg/kg/day - 10 male and 10 female rats
40 mg/kg/day - 10 male and 10 female rats
80 mg/kg/day - 10 male and 10 female rats
160 mg/kg/day - 10 male and 10 female rats

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Clinical observations were recorded weekly.

BODY WEIGHT: Yes
Animals were weighed at the start of the study, weekly thereafter, and at necropsy.

HAEMATOLOGY: Yes
Hematology parameters included hematocrit, hemoglobin concentration, erythrocyte count, reticulocyte count, nucleated erythrocyte count, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, leukocyte count and differential, methemoglobin concentration, and Heinz body count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:after 3 and 23 days in rats and at study termination (Day 93) in rats
- Animals fasted:No data
- How many animals:20
- Parameters examined:Urea nitrogen, creatinine,total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile salts.

OTHER:
Survival of species :Yes
Organ Weight : Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Complete necropsies were performed on all animals.During each necropsy, all tissues were examined in situ for gross lesions. At study termination, selected organ weights (spleen, liver, thymus, heart, lung, and right testis and kidney) were determined.

Extra rats (l0 sex/dose group) were included for clinical pathology evaluations that were performed on study days 3 and 23; after the second blood collection, these animals were terminated and discarded without examination.

HISTOPATHOLOGY: Yes
Histopathologic evaluations were performed on all animals in the vehicle control and 160 mg/kg groups. Tissues routinely examined.
Tissues examined in lower dose groups included the bone marrow,kidneys, liver, and spleen in rats.
Tissue examined microscopically in all control,high dose, and early death animals included adrenal glands, brain, clitoral glands, esophagus, bone marrow (femur), gallbladder (mice), heart, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum),kidneys, liver, lungs and mainstem bronchi, lymph nodes (mandibular, mes-enteric), mammary gland, ovaries, pancreas, parathyroid glands, pituitary gland, preputial glands, prostate gland, salivary glands, seminal vesicles, spinal cord, spleen, stomach, testis (with epididyrnis), thymus, thyroid gland, trachea,urinary bladder, uterus, and any gross lesions seen at necropsy.

Statistics:

In-life data (body weights, clinical observations) and microscopic findings were collected and summarized using a computerized system.Body weight and organ weight data were analyzed using the parametric comparison procedures of or Dunnett.Clinical pathology data were analyzed using the nonparametric comparative procedures of Shirley or Dunn. The Fisher exact test, a procedure based on the overall proportion of affected animals, was used to analyze histopathology findings , On all tables, values are expressed as group mean and SE.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of toxicity was observed.Blue discoloration (cyanosis) of the skin in areas easily observed (ear, nasal,genital, and footpad regions).Survival was similar between rat treatment groups receiving equivalent dose of m-CA and control groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

Clinical signs of toxicity was observed.Blue discoloration (cyanosis) of the skin in areas easily observed (ear, nasal,genital, and footpad regions).Survival was similar between rat treatment groups receiving equivalent dose of m-CA and control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

m-CA treatment produced a 10.5% depression in group mean body weight relative to control in the 160 mg/kg male rat dose group.

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

Description (incidence and severity):

Rats in the higher dose groups (80 and 160 mg/kg) were found to have darker eyes (iris) than control.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A dose-related increase in blood methemoglobin concentration was detected in all m-CA treatment groups.At Days 23 & 93, the methemoglobin increase wasalso greater in rats given m-CA.At each dose, the methemoglobin response was greater in female rats.

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

Description (incidence and severity):

Treatment produced a dose-related increase in relative spleen weight.A similar trend ocurred in absolute spleen weight.The only other treatment related change was a slight increase in relative heart weight in rats given 80 and/or 160 mg/kg m-CA.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Splenomegaly was observed in both rat sexes at a lower dose of m-CA (40 mg/kg).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Splenic vascular congestion and cellular infiltration/fibrosis of the splenic capsule were microscopic lesions found only in rats (both sexes).

Histopathological findings: neoplastic:

not specified

Details on results:

- HAEMATOLOGY :
*A dose-related increase in blood methemoglobin concentration was detected in all m-CA treatment groups.At Days 23 and 93, the methemoglobin increase was also greater in rats given m-CA.Male rats given 160 mg/kg and female rats given 80 and 160 mg/kg m-CA showed high and relatively constant (sustained) methemoglobin concentrations at each sampling time, suggesting a maximal response had been achieved.At each dose, the methemoglobin response was generally greater in female rats.
*Exposure to m-CA produced dose-related decreases in erythrocyte count, which were statistically significant at the higher doses.
*Anemia was associated with a decrease in hematocrit in rats.At study termination, Heirlz body formation in erythrocytes in association with decreased hemoglobin, hematocrit, and red blood cell count was a prominent treatment-related effect.
*The percentages of erythrocytes containing Heinz bodies : With m-CA, rats at the 40 mg/kg dose level and above showed significant and dose-related increases.

- CLINICAL SIGNS AND MORTALITY : Clinical signs of toxicity was observed.Blue discoloration of the skin in areas easily observed (ear, nasal,genital, and footpad regions). These effects were attributed to anoxia resulting from the formation of high levels of methemoglobin.


- OPHTHALMOSCOPIC EXAMINATION : Rats in the higher dose groups (80 and 160 mg/kg) were found to have darker eyes (iris) than control.

- HISTOPATHOLOGY : Microscopic lesions consisted of excess hematopoietic activity in the bone marrow, spleen and/or liver, and deposition of hemosiderin pigment in the bone marrow, spleen, liver Kupffer cells), or kidney cortex (rats). These changes were seen more frequently and/or severely in rats . Sex differences were not evident in lesion frequency/severity.Splenic vascular congestion and cellular infiltration/fibrosis of the splenic capsule were microscopic lesions found only in rats (both sexes).Splenic congestion,cellular infiltration/fibrosis was found in m-CA-treated rats.

Dose descriptor:

LOAEL

Effect level:

10 other: mg/kg/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects observed on the following parameters examined : Body weight Organ weight Haematology Ophthalmoscopic examination Organ Weight Histopathology

Critical effects observed:

not specified

Conclusions:

In a repeated dose toxicity study of 3-chloroaniline, LOAEL was considered to be 10 mg/kg bw/day when administered orally to rats.

Executive summary:

The subchronic study was conducted to evaluate the toxic effects of repeated administration of 3-chloroaniline to male and female F344 rats by gavage was evaluated. m-CA was administered to 10 animals/sex/species in deionized water at dosages of 0, 10, 20, 40, 80, and 160 mg/kg for 13 weeks. Blood samples for clinical pathology were collected after 3 and 23 days in rats and at study termination (Day 93) in rats.No mortalities occurred that could be directly attributed to treatment.Transient clinical signs of toxicity observed after dosing included cyanosis in rats.Methemoglobin formation was directly related to dosage (rats) and duration of treatment (rats). At study termination, Heirlz body formation in erythrocytes in association with decreased hemoglobin, hematocrit, and red blood cell count was a prominent treatment-related effect. Enlarged spleens (gross necropsy observation) and increased spleen weight were treatment effects. Microscopic lesions typical of increased red blood cell production were found in hematopoietic tissues (bone marrow, spleen, and liver), while lesions due to increased red cell destruction were found in these tissues and also the kidneys (rats).Sex differences in lesion incidence/severity were not evident.

Therefore,the lowest observed adverse effect level (LOAEL) for repeated dose toxicity study was considered to be 10 mg/kg/day for the 13-weeks study, it is regarded that there is no repeated dose toxicity at concentrations lower than 10 mg/kg bw/day when administered orally. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The data is K2 level as the data has been obtained from the experimental study from the reliable journal ‘Toxicology'.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Principles of method if other than guideline:

A subacute study was conducted to evaluate the toxic effect of repeated administration of 3-chloroaniline to rats by the inhalative route.

GLP compliance:

not specified

Species:

rat

Strain:

not specified

Sex:

not specified

Route of administration:

inhalation

Type of inhalation exposure:

not specified

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

14 Days

Remarks:

Doses / Concentrations:
8.800 mg/kg bw/d
Basis:

Control animals:

not specified

Observations and examinations performed and frequency:

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

OTHER: Organ Weight : Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

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:

effects observed, treatment-related

Description (incidence and severity):

erythrocytes,extrame dullary haematopoiesis,haematocrit,haemoglobin,methaemoglobinaemia

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Changed Enzyme activity

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

weight increased in spleen

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

congestion observed in spleen

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

haematopoiesis and haemosiderosis in spleen

Histopathological findings: neoplastic:

not specified

Dose descriptor:

LOEL

Effect level:

8.8 other: mg/kg bw/d

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Effects observed on clinical chemistry,haematology,organ weights,gross and histopathology.

Critical effects observed:

not specified

Conclusions:

In a repeated dose toxicity study of 3-chloroaniline, LOEL (Lowest onserved effect level) was considered to be 8.800 mg/kg bw/d when administered by inhalative route to rat.

Executive summary:

In a repeated dose toxicity study, the toxic effects of repeated administration of 3-chloroaniline to rat by the inhalative route was evaluated. Rats were given 3-chloroaniline for 14 days at a dose concentration of 8.800 mg/kg bw/day. Changed Enzyme activity, in blood -erythrocytes,extramedullary haematopoiesis,haematocrit,haemoglobin and methaemoglobinaemia observed,as well as in spleen congestion,haematopoiesis,haemosiderosis and weight increased was observed.Therefore,LOEL(Lowest onserved effect level) for repeated dose toxicity study was considered to be 8.800 mg/kg bw/day for the 14 days study,it is regarded that there is no repeated dose toxicity at concentrations lower than 8.8 mg/kg bw/day when administered by inhalative route.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

8.8

Study duration:

subacute

Species:

rat

Quality of whole database:

The data is K4 level.The data has been obtained from the experimental study from the reliable 'REPDOSE: A database on repeated dose toxicity studies of commercial chemicals—A multifunctional tool'.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity (Oral)

WoE Summary of 108-42-9 for repeated dose toxicity

Based on the various studies available with Klimish rating 2 and 4 for the target as well as the read across substances for CAS: 108-42-9 based on the category approach of organic functional group along with similar mechanistic approach and having structural similarities defined by QSAR toolbox.

The results for target as well as analogues are summarized as follows

Sr. No	End point	Value	Species	Route	Effects	Remarks
1	LOEL	185 mg/kg bw/day (nominal)	Rat	Oral (gavage)	Effects observed on Body weight, Food consumption and Relative organ weight	Predicted data for CAS: 108-42-9
2	LOAEL	10 mg/kg/day	Rat	Oral (gavage)	Effects observed on the following parameters examined :Body weight, Organ weight, Haematology, Ophthalmoscopic examination, Organ Weight, Histopathology	Data from publication for CAS: 108-42-9
3	LOAEL	10 mg/kg/day	Mouse	Oral (gavage)	Effects observed on the following parameters examined :Body weight, Organ weight and Haematology	Data from publication for CAS: 108-42-9
4	TDLo - Lowest published toxic dose	10000 mg/kg	Rat	Oral	Formation of methemoglobinemia was observed.	Data from publication for CAS: 108-42-9
5	TDLo - Lowest published toxic dose	6370 mg/kg	Rat	Oral	Changes in spleen weight, changes in erythrocyte (RBC) count, Enzyme inhibition, induction, or change in blood or tissue levels was observed.	Data from publication for RA CAS: 95-76-1

 

Based on the studies summarized in the above table with oral routes it can be observed that the low observed effect value varies (LOEL and LOAEL) from 10 mg/kg/d to 185 mg/kg bw /d based on the predicted data as well as data from publication for target substance. Also the lowest published toxic dose value for target and read across is TDLo = 6370 mg/kg to 10000 mg/kg. The effect observed on the above doses are-

·      Resulted in decreased body weight, decreased relative organ weight as well as decreased food consumption.

·      Effects observed on the following parameters examined : Body weight, Organ weight, Haematology, Ophthalmoscopic examination, Organ Weight, Histopathology

·      Formation of methemoglobinemia was observed.

·      Changes in spleen weight, changes in erythrocyte (RBC) count, Enzyme inhibition, induction, or change in blood or tissue levels was observed.

Thus based on the above results it can be concluded that substance CAS: 108-42-9 is expected to show the similar toxicological effect based on the effects observed on the other category members. Since the low effective dose value (LOEL and LOAEL) is greater than 10 mg/Kg/day thus based on this value it can be concluded that substance CAS: 108-42-9 is considered to be not toxic to repeated dose via oral route below the dose level of 10 mg/Kg bw/d. There are no known evidence of adverse effect to human of CAS: 108-42-9. Based on this value it can be concluded that CAS: 108-42-9 is classified as STOT RE 2 category as the criteria of CLP regulation.

 

Repeated dose toxicity (Inhalation)

WoE Summary of 108-42-9 for repeated dose toxicity

Based on the various studies available for the target substances for CAS: 108-42-9 .The results for target substance are summarized as follows-

Sr. No	End point	Value	Species	Route	Effects	Remarks
1	LOEL	8.8 mg/kg bw/d	Rat	Inhalation	Changed Enzyme activity, in blood -erythrocytes, extramedullary haematopoiesis, haematocrit,haemoglobin and methaemoglobinaemia observed, as well as in spleen congestion, haematopoiesis, haemosiderosis and weight increased was observed.	Data from publication for CAS: 108-42-9
2	LOEL	44 mg/kg bw/d	Rat	Inhalation	Clinical symptons of cyanosis was observed.	Data from publication for CAS: 108-42-9
3	LOEL	72.875 mg/kg bw/d	Rat	Inhalation	In eye opacity was observed, liver weight increased as well as spleen atrophy was observed.	Data from publication for CAS: 108-42-9

 

Based on the studies summarized in the above table with inhalation routes it can be observed that the low observed effect value varies (LOEL) from 8.8 mg/kg bw/d to 72.875 mg/kg bw/d based on the publication data for target substance. The effect observed on the above doses are-

·  Changed Enzyme activity, in blood -erythrocytes, extramedullary haematopoiesis, haematocrit, haemoglobin and methaemoglobinaemia observed, as well as in spleen congestion, haematopoiesis, haemosiderosis and weight increased was observed.

· Clinical symptons of cyanosis was observed.

· Formation of methemoglobinemia was observed.

·  In eye opacity was observed, liver weight increased as well as spleen atrophy was observed.

Since the low effective dose value (LOEL) is greater than 8.8 mg/kg bw/d thus based on this value it can be concluded that substance CAS: 108-42-9 is considered to be not toxic to repeated dose via inhalation route below the dose level of 8.8 mg/kg bw/d. There are no known evidence of adverse effect to human of CAS: 108-42-9. Based on this low value it can be concluded that CAS: 108-42-9 is classified as STOT RE 2 category as the criteria of CLP regulation.

 

Repeated dose toxicity (dermal)

In accordance with column 1 of Annex IX, repeated dose toxicity by the dermal route was considered for waiver as the data for acute toxicity by the dermal route has already been provided as part of Annex VIII information requirements.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

In a repeated dose toxicity study of 3-chloroaniline, LOAEL was considered to be 10 mg/kg bw/day when administered orally to rats.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

In a repeated dose toxicity study of 3-chloroaniline, LOEL (Lowest onserved effect level) was considered to be 8.800 mg/kg bw/d when administered by inhalative route to rat.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

In accordance with coloumn 1 of Annex IX, repeated dose toxicity by the dermal route was considered for waiver as the data for acute toxicity by the dermal route has already been provided as part of Annex VIII information requirements.

Justification for classification or non-classification

The substance shows repeated dose toxicity effect for oral and inhalation route and thus will be considered for further classification in STOT RE 2 category as per CLP regulation.