Paraffin waxes and Hydrocarbon waxes, chloro

Endpoint summary

Description of key information

13 -Week Dietary Toxicity Study in Rats

13 -Week Oral (Gavage) Toxicity Study in Rats

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet: ad libitum
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The appropriate amount of the test material was ground with 1000 grams of Certified Rodent Chow #5002.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

100 gram samples from the top, middle, and bottom of the batches were tested for homgeneity of test article dispersion prior to use.

Duration of treatment / exposure:

13-weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
100, 900, 3750 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked included: signs of overt toxicity, morbidity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed examination took place before the study was terminated.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on days 0 and 5.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Daily

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, All rats received a post mortem examination at the termination of the study.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

All mortalities were due to blood sampling procedures

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slightly reduced mean body weight gain in 3750 mg/kg/day males (high concentration of non-nutritive test article may have influenced).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

indications of increased mean food consumption at 3750 mg/kg/day (high concentration of non-nutritive test article may have influenced).

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

slightly elevated liver transaminase levels at 3750 mg/kg/day (females)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

A general tendency of reduced urine volume with corresponding increases in specific gravity at 3750 mg/kg/day (particularly for males).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

liver weight increase at 3750 mg/kg/day (females)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

hypertrophy and cytoplasmic fat vacuolation in liver at 3750 mg/kg/day

Details on results:

There were no significant changes in appearance or behavior. Four deaths, including 2 control females, occurred following blood sample collection and were not considered treatment related.

There was evidence of an effect on the liver at 3750 mg/kg/day with slight changed in alanine and aspartate aminotransferase and alkaline phosphate levels, increased liver weights, oil red "O" staining, cytoplasmic fat vacuolation and hepatocellular hypertrophy.

Key result

Dose descriptor:

NOAEL

Effect level:

900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Dose descriptor:

LOAEL

Effect level:

3 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

3 750 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The NOAEL from this study is 900 mg/kg/day for both male and female rats. Effects, primarily in the liver, were noted at 3750 mg/kg/day.

Executive summary:

The NOAEL from this study is 900 mg/kg/day for both male and female rats. Effects, primarily in the liver, were noted at 3750 mg/kg/day.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet: ad libitum
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Rats were gavaged daily for 13-weeks

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

25 mL samples of the prepared test solutions were collected and analyzed for test article concentration prior to adminstration at weeks 1, 4, 8, and 12.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
100, 900, 3750 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

This study also included the dosing of a radio-labelled CP during several points during the study. Details are discussion in Section 7.1.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, 7 days a week
- Cage side observations checked included: signs of overt toxicity, morbidity and mortality.

Sacrifice and pathology:

All rats received a post mortem examination at the termination of the study. Each rat was subjected to a gross examination and the weights of key organs, including the liver, kidney(2), thymus, spleen, adrenal (2), thyroid (2), testis (2), ovary (2), brain and pituitary, were recorded. Five rats (randomly selected) from each sex/dose were examined for hepatic microsomal parameters. Hematoxylin and eosin stained paraffin sections of most tissues were examined for the high does and control animals. In addition, liver section were examined for all dose groups.

Statistics:

Microscopic examination was carried out on the liver, both kidneys, spleen and any other organ that had displayed a gross lesion. Body weights, food consumption, food conversion ratios and absolute and relative organ weights were subjected to statistical analysis by comparing treated groups with their respective controls. Comparison was by analysis of variance and an appropriate "t-test". The results from the hepatic microsomal determination were compared using the Mann-Whitney U-test.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

several mortalities occurred in study, but were attributed to handling/dosing errors

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slight increase at top dose

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

slightly increased liver weights (females only)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

dose-related evidence of liver changes (females only), characterized as granulomatous inflammation, necrosis and positive fat staining. The significance of an increased incidence of nephrosis and interstitial pneumonia in high dose male animals was described as obscure.

Details on results:

Inflammatory changes and necrosis in the liver of female rats occurred at all doses, with increased intensity of Oil-red O staining. Mean absolute and relative organ weights were also increased.

Dose descriptor:

NOAEL

Effect level:

3 750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects were observed at the highest dose tested

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Conclusions:

There were no treatment related adverse effects to the male rats and the NOAEL was established at the top dose (3750 mg/kg/day). Effects in female rats, primarily in the liver, were observed at all dose levels and thus a LOAEL was established at 100 mg/kg/day.

Executive summary:

There were no treatment related adverse effects to the male rats and the NOAEL was established at the top dose (3750 mg/kg/day). Effects in female rats, primarily in the liver, were observed at all dose levels and thus a LOAEL was established at 100 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

900 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are repeated dose subchronic and chronic rodent toxicity studies available for both liquid and solid grade LCCPs. No toxicity of LCCP was observed in male or female mice in 13-week studies or in the 2-year studies. In rats, a dose-related inflammation of the liver was observed in the 13-week studies and in the 2-year studies. This effect was most evident in female rats administered LCCP by bolus gavage, and occurred at doses of 100 mg/kg/day and greater.  Dietary administration of LCCP produced less severe liver effects at significantly higher doses. The mechanism(s) by which the rat liver effect occurs are not known, and the reasons for the much greater sensitivity of the female rat to bolus gavage doses, and for the remarkable species difference between rats and mice are not understood.  Based upon the available studies the LOAEL for effects in the female rat liver was 100 mg/kg/day following gavage administration, and the NOAEL for effects in male and female rat liver was 900 mg/kg/day following dietary administration.

The US National Toxicology Program (NTP, 1986) studied the subchronic and chronic oral (gavage) toxicity of a liquid grade LCCP (C23 average, 43% chlorine) in rats and mice. The subchronic oral (gavage) toxicity of a similar liquid LCCP (C22-26, 43 % Chlorine) was studied in rats by IRDC, 1984a. The subchronic oral (dietary) toxicity of a solid grade (C22-28, 70% Chlorine) was also studied in rats by IRDC, 1984b.

Liquid Grade LCCP – oral gavage studies

NTP, 1985 conducted chronic repeated dose toxicology studies of a liquid grade LCCP (C23, 43% chlorine), by administering the substance in corn oil by gavage to groups of 50 F344/N rats and 50 B6C3Fl mice of each sex, 5 days per week for 103 weeks. Additional groups of 10 rats per sex and dose were examined at 6 and at 12 months. Male rats received doses of 0, 1,875, or 3,750 mg/kg body weight; female rats were given 0, 100, 300, or 900 mg/kg. Male and female mice received 0, 2,500, or 5,000 mg/kg. Doses selected for the 2-year studies were based on the results from 13-week studies in which rats of each sex received 0 to 3,750 mg/kg, and mice of each sex, 0 to 7,500 mg/kg. No toxicity of chlorinated paraffins (C23, 43% chlorine) was observed in male or female mice in the 13-week studies or in the 2-year studies. In contrast to the absence of chemically related toxicity in mice, a dose-related inflammation of the liver was observed in female rats (but not in male rats) in the 13-week studies and in male and female rats at 6 and 12 months in the 2-year studies. A no-observed-effect-level was not established for this effect. The chronic LOAEL for effects in the liver was 1875 mg/kg bw/day in male rats and 100 mg/kg bw/day in female rats. The NOAEL for chronic toxicity in mice was 5,000 mg/kg/day.

IRDC, 1984a conducted a 13-week repeated dose toxicology study of a liquid grade LCCP (C22-26, 43 % Chlorine) . The test substance was similar to that studied by NTP and the study was conducted in accordance with GLPs. The test substance was administered in corn oil by gavage daily for 13 weeks to groups of fifteen Fischer rats of each gender at dose levels of 0, 100, 900 or 3,750 mg/kg/day. As seen in the NTP study, liver effects were observed in female rats. There was evidence of liver toxicity in females of all treated groups (increased absolute and relative liver weight, granulomatous inflammation, necrosis, and positive fat staining. There were no effects in the livers of treated male rats, although trace/mild nephrosis was reported in male rats receiving 3750 mg/kg/day. The NOAEL for male rats from this study was 900 mg/kg bw/day and the LOAEL for female rats was 100 mg/kg bw/day (no NOAEL could be established). 

In summary, the following repeated dose effect levels are established for liquid LCCP administered by gavage:

	Subchronic		Chronic
	NOAEL	LOAEL	NOAEL	LOAEL
Mice (male and female)	7,500 (highest dose tested), NTP		5000 (highest dose tested), NTP
Rat (male)	900 (NOEL, kidney), IRDC 3,750 (highest dose tested, NTP)	3750 (LOEL, kidney), IRDC	Not established	1875 (liver, lowest dose tested), NTP
Rat (female)	Not established	100 (liver, lowest dose tested), NTP, IRCD	Not established	100 (liver, lowest dose tested), NTP

All values shown are mg/kg/day.

The reasons for the sensitivity of the female rat liver to liquid LCCP effects following gavage administration and the relevance of these findings to humans are not understood. Similar effects were not seen at much higher doses in male rats and male and female mice. Furthermore, similar studies of shorter chain chlorinated paraffins showed them to be generally more toxic in rats, but did not produce this type of liver effect.

Solid Grade LCCP – dietary study

IRDC, 1984b also conducted a 13-week repeated dose toxicology study of a solid grade LCCP containing higher levels of chlorine (C22-28, 70% Chlorine). The test design was identical to that used for the liquid grade LCCP study except that the test substance was administered in the diet, and the study was conducted in accordance with GLPs. The LCCP solid product was administered for 13-weeks via the diet to groups of 15 Fischer rats of each gender at concentrations resulting in dose levels of 0, 100, 900 or 3,750 mg/kg/day. To achieve these dose levels initial concentrations of solid LCCP product in the diet were 1000, 9000 and 37,500 ppm in the low-, mid-, and high-dose groups respectively. To sustain the desired doses as the animals grew, these concentrations were increased throughout the study. Week 13 concentrations for the low-, mid- and high-dose group males were approximately 1682, 14,525 and 57,386 ppm, respectively. Week 13 concentrations for the low-, mid- and high-dose group females were approximately 1393, 11,649 and 46,997 ppm, respectively. There were no treatment-related mortalities or clinical signs seen in this study. Increased food consumption was noted at the high dose level, possibly due to nutritional displacement caused by the high concentrations of test substance in the diet (up to 5.8%). High dose male animals showed slightly reduced body weight gain. There was evidence of a mild effect on the liver in the high-dose animals only. This was characterized by slightly elevated liver enzyme levels in serum, increased liver weights, hepatocellular hypotrophy, and cytoplasmic fat vacuolation. The microscopic changes were more prominent among females. Liver microsomal determinations were generally consistent with these findings. The liver inflammation and necrosis observed in studies of liquid grade LCCPs were not seen in this study. One important methodological difference was bolus gavage dosing of the liquid LCCPs.

 

For this 70% chlorination solid LCCP product, effects in the liver occurred only at a very high exposure level of 3,750 mg/kg/day (concentrations in the diet up to 5.8%). Based on the findings of this study, a NOAEL of 900 mg/kg/day was identified for male and female rats based on the observation of slight liver effects at the higher dose level of 3,750 mg/kg/day. These values are considered to be the most relevant for evaluating human safety.

Justification for classification or non-classification

Justification for Classification or Non-Classification

Based upon a weight-of-evidence evaluation of the available data no repeated dose toxicity classification is assigned to LCCPs. Key factors underlying the justification for no classification include the following: A 90-day repeated dose NOAEL of 900 mg/kg/day (oral dietary, male and female rat) has been established for solid grade LCCP, which does not require classification. 90-day and 2-year repeated dose NOAELs of 7,500 and 5,000 mg/kg/day, respectively, have been established for liquid grade LCCPs in mice, which do not require classification. 90-day repeated dose NOAELs of 3,750 and 900 mg/kg/day were established for similar liquid grade LCCPs in separate studies of male rats, which do not require classification. Liquid LCCPs have been shown to produce liver inflammation in female rats following bolus gavage dosing for 90-days or 2-years, and in male rats following bolus gavage dosing for 2 years. This effect occurred at the lowest doses tested (1,875 mg/kg/day for males; 100 mg/kg/day for females). The mechanism(s) by which this effect occurs are not known, nor are the reasons for the much greater sensitivity of the female rat to gavage doses and the remarkable species difference between rats and mice. The relevance of this finding to humans are not understood, although the dietary results are considered to be more relevant to the human exposure situation. Therefore, consideration of the full body of available evidence does not justify classification of LCCPs for repeated dose toxicity.