Maleic anhydride

Endpoint summary

Description of key information

Suitable data were available to assess the toxicity of maleic anhydride after repeated dose exposure. Therefore, the available data from repeated dose toxicity studies conducted with maleic anhydride were used in a weight of evidence approach to assess the specific target organ toxicity of the target substance. Based on the results and in accordance with ATP13 to CLP Regulation 1272/2008 classification as STOT RE 1, H372 is warranted.

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

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

GLP compliance:

no

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 - 9 months
- Housing: in pens according to sex and dose
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 months

Route of administration:

oral: feed

Vehicle:

peanut oil

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Storage temperature of food: The basal ration consisted of ground laboratory chow supplemented with 1% peanut oil. A premix composed of 5% maleic anhydride and the basal ration was used to prepare the test diets

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the premix by a gas chromatographic method confirmed the concentration and stability of the chemical in feed.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a preliminary tolerance study, beagle dogs fed maleic anhydride in their diets at a concentration that provided 100 mg/kg/day showed a considerable decrease in food intake along with a loss of body weight within one week. Dogs fed a dose of 20 mg/kg/day for 2 weeks, then 40 mg/kg/day for 2 weeks, and finally 60 mg/kg/day for 2 weeks showed no decrease in food consumption, body weight or other signs of toxicity. Based on these findings, dose levels of 20, 40 and 60 mg/kg/day maleic anhydride were selected for the 90-day study.

Positive control:

no

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: Yes (for each pen of four dogs twice a week) and the average food intake/dog/day was calculated

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the beginning of the study and after 22 and 83 days of treatment
- How many animals: all animals
- Parameters checked: packed cell volume (PCV), erythrocyte count (RBC), hemoglobin concentration (Hgb), total and differential leukocyte counts (WBC), and microscopic examination of erythrocytes.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to beginning of the study, on day 26 of treatment and just prior to termination of the study (see below)
- Parameters checked: Blood urea nitrogen (BUN), serum alkaline phosphatase (AP), serum glutamic oxalacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT) activities (prior to initiation of the study and after 26 and 83 days on test). Because of some aberrant values for AP, SGOT, and SGPT for female control dogs, the determinations were repeated on this group of dogs on day 90 of treatment. Glucose was measured in serum from all dogs after 26 and 83 days on test. Phenolsulphthalein excretion (PSP) was determined on control male dogs and those receiving 60 mg/kg/day maleic anhydride for 85 days.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were obtained from each dog by catheterization prior to the beginning of the study and after 25, 76, and 85 days on the tests.
- Parameters checked: specific gravity, pH, sugar, albumin, ketones, occult blood and bilirubin, and the urine sediment was examined microscopically. The same determinations were made on urine samples obtained at necropsy from the urinary bladder of each dog by means of a needle and syringe.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Body weights, food consumption, organ weights, organ to body weight ratios and hematological and clinical chemistry data were analyzed statistically by an analysis of variance and Dunnett's test. The level of significance selected for all cases was p<0 .05.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For futher details, see section below "Details on results".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For futher details, see section below "Details on results".

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

For futher details, see section below "Details on results".

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

For futher details, see section below "Details on results".

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

BODY WEIGHT AND WEIGHT GAIN: Female dogs in all 20 dose groups were slightly but not significantly heavier than controls at the beginning of the study and remained so throughout the experiment. While females in the 60 mg/kg/day groups were statistically significantly slightly heavier than controls at the beginning of the study and remained so throughout the experiment.

FOOD CONSUMPTION AND COMPOUND INTAKE: Food intake was decreased for the first few weeks in both sexes of dogs receiving 60 mg/kg/day (lower mean food consumption values than the controls or their own pretest levels). For the remainder of the study,food intake was similar for the control dogs and those receiving 60 mg/kg/day maleic anydride. This transient effect was not considered to be "adverse".

HAEMATOLOGY: After 22 days of treatment, the mean PCV of male dogs receiving 60 mg/kg/day maleic anhydride was 4 % lower than the value obtained prior to the beginning of the study. However, there was a greater depression for the same parameter in controls. After 83 days of treatment, the mean PCV of the male dogs receiving 60 mg/kg/day was depressed further while among the controls, the mean PCV had increased somewhat over that found after 22 days of treatment. At 83 days, the mean PCV values for these two groups of dogs were significantly different. The mean Hgb concentration of male dogs fed 60 mg/kg/day for 83 days was slightly lower than the control mean, the difference being one of borderline statistical significance. (Both, the mean PCV and HgB values of the male dogs that received 60 mg/kg/day for 83 days are within two standard deviations of the mean values obtained recently for 64 control male beagle dogs and therefore considered not to be biologically significant).

ORGAN WEIGHTS: The significant increase in the mean absolute weight of the kidneys of female dogs fed the 60 mg/kg/day dose is considered to be due to the fact that these dogs were larger than the control dogs and thus had larger kidneys.

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Transient reduction of food consumption at the beginning of the study , and minor changes of blood parameters in male dogs were not considered adverse

Critical effects observed:

not specified

Conclusions:

In a subchronic oral study, transient test item related effects on food consumption in both sexes and blood parameters in males were observed. Based on these results, the NOAEL is considered to be 60 mg/kg bw/day.

Executive summary:

In a subchronic toxicity study (similar to OECD Guideline 409) maleic anhydride, (purity >99.5%) was administered to 4 Beagle dogs/sex/dose in diet at dose levels of 0, 20, 40 or 60 mg/kg bw/day.

 

Food intake was decreased for the first few weeks in both sexes of dogs receiving 60 mg/kg/day (lower mean food consumption values than the controls or their own pretest levels). For the remainder of the study, food intake was similar for the control dogs and those receiving 60 mg/kg bw/day maleic anydride. This transient effect was not considered to be "adverse". All female dogs in 20 mg/kg bw/day dose group were slightly but not significantly heavier than controls at the beginning of the study and remained so throughout the experiment. While females in the 60 mg/kg bw/day groups were statistically significantly slightly heavier than controls at the beginning of the study and remained so throughout the experiment.Haematological studies conducted after 83 days of exposure indicated equivocal decreases in packed cell volume and haemoglobin concentration of male dogs receiving 60 mg/kg bw/day. This effect was not considered to be "adverse". No other changes were observed that were considered relatable to the ingestion of diets containing maleic anhydride.

 

The NOAEL is  60 mg/kg bw/day.

This subchronic toxicity study in the dog is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 409) in dogs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subchronic

Species:

dog

Quality of whole database:

comparable to guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

Evaluation of maleic anhydride toxicity in rats, hamsters and monkeys after 6 months of inhalation administration.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Weight at study initiation: 230-310 g (males); 174-200 g (females)
- Age at study initiation: about 8 weeks
- Housing: single
- Diet: ad libitum (except during exposure)
- Water: ad libitum (except during exposure)
- Acclimation period: at least 2 weeks

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 15 m² cubical stainless-steel and glass chambers with pyramidal tops
- System of generating particulates/aerosols: Atmospheres containing the test material were generated by heating maleic anhydride, which has a melting point of 53°C, and by transporting vapors from the melt to the chambers with a stream of nitrogen gas. The overall quantity af maleic anhydride vaporized was determined by weighing the "boat" containing maleic anhydride prior to exposure and upon recrystallization of the compound after exposure.


TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations were sampled at least three times per day
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations in the chamber (total maleic; i .e ., maleic anhydride plus maleic acid) were monitored by drawing samples through Tenax columns and by quantifying the retained material, after thermal desorption into a nitrogen steam, using a gas chromatograph equipped with a flame ionization detector and a stainless-steel column packed with 1 .5% OV-l01 on 100-120 Chromosorb G-HP. The range of column and detector temperatures was 115 to 150°C and 205 to 220°C, respectively.

Duration of treatment / exposure:

6-months / 132 or 133 exposures

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

0 mg/m³ air (analytical)

Dose / conc.:

1.1 mg/m³ air (analytical)

Dose / conc.:

3.3 mg/m³ air (analytical)

Dose / conc.:

9.8 mg/m³ air (analytical)

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: 16- hour fasting period

Positive control:

no

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before and after each exposure

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week for the initial 2 months and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: monthly intervals
- Dose groups that were examined: on all test animals.

HAEMATOLOGY: Yes
- Animals fasted: Yes
- How many animals: 5 rats/sex/group from the control and high-exposure groups at 3 months and 5 rats/sex/group at 6 months
- Parameters checked: hemoglobin, hematocrit, total erythrocyte count, and total and differential leucocyts count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 3 and 6 months
- How many animals: 5 rats/sex/group from the control and high-exposure groups at 3 months and 5 rats/sex/group at 6 months
- Parameters checked: glucose, urea nitrogen, serum glutamic pyruvic transaminase activity, serum alkaline phosphatase activity, carbon dioxide, erythrocyte and plasma cholinesterase activity, and terminal brain cholinesterase activity

URINALYSIS: Yes
- Time schedule for collection of urine: 5 rats/sex/group from the control and high-exposure groups at 3 months and 5 rats/sex/group at 6 months
- Metabolism cages used for collection of urine: Yes
- Parameters checked: volume, pH, specific gravity, description of color and appearance, qualitative tests for albumin, glucose, bilirubin, ketones and occult blood and microscopic examination of the sediment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (on the control and high-exposure groups)

Statistics:

All statistical analyses compared the treatmant groups with the control group, by sex. Not all parameters were statistically evaluated at all intervals. Some parameters at some intervals were evaluated statistically after a review of the data indicated that a possible exposure related effec may have been present. The hematological, biochemical and urinalysis parameters (terminal) and absolute and relative organ weights (terminal) were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie using Dunnett's multiple comparison tables to judge significance of differences.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

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:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: Survival was >90% in all groups. Nasal and ocular irritations were observed at all maleic anhydride-exposed animals during the study. High-exposure rats exhibited a red-tinged nasal discharge, isolated cases of ocular discharge and sneezing. These effects were less severe in animals from the low- and mid-exposure groups.

BODY WEIGHT AND WEIGHT GAIN: Statistically significant reductions in body weights were observed in the male rats in the mid-exposure group on days 78, 92, 106, and 127; and in the high-exposure group on days 40 through 189 (p<0.05). Statistically significant reductions in body weights were observed in the female rats in the mid-exposure groups on days 54, 127, 141, 148, 155, an 156; and in the high-exposure group on days 37, 47, 57, 71, 85-176, and 189 [<10%, (p<0.05)]. However, at the end of the treatment period, terminal body weights were reduced only for male rats from the high-exposure group. There were no statistically significant effect on body weight at the low-exposure group for either sex of rat.

HAEMATOLOGY: significant decrease in monocyte count in high dose male rats; no biological significance was attributed to these values since they were within the normal range af variation

CLINICAL CHEMISTRY: Clinical chemistry results indicated a few differences which were statistically different from the control values; however, none of these differences were considered to be treatment related or consistent in both sexes. These differences were:
- glucose was reduced in males (- 9%) and increased in females (+ 17%) of the mid dose group after 6 months while both sexes of the high dose group had reduced values after 3 months (-18% and -21%, respectively);
- serum CO2 levels were slightly elevated in the high dose males after 6 months (+ 11%);
- urea nitrogen was slighly increased in high dose females after 6 months (+ 26%);
- red blood cell cholinesterase activity was reduced in mid dose males at 6 months (-17%) and brain cholinesterase was increased in mid (+ 27%) and high dose males (+ 21%) at 6 months

URINALYSIS: Female rats in the low-dose group had a significantly reduced urine volume (- 59%) with an increased specific gravity (+ 3%). These observations were not dose related and thus not considered related to treatment.

ORGAN WEIGHTS: Organ weight changes were observed in rats (8 of 9 tissues weighed). The only changes present in both the mid- and high-exposure groups were increased relative pituitary weight in female rats (+ 23%/+ 30%), increased relative adrenal weight (females: + 28%/36%; males: + 19% in the high-exposure group) and reduced absolute (- 26%/- 28%) and relative thyroid weight (- 24%/- 20%) in male rats. Since the histopathology data did not provide evidence of tissue damage, these changes were not considered to be related to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC: Hyperplastic changes in the nasal tissues, which ranged in grade of severity from trace to mild, were present in all maleic anhydride exposed rats. Both the incidence and grade appeared to be exposure-related. Metaplastic changes in the nasal tissues were present in rats at all exposure levels and the incidence increased in a nonlinear fashion. Rats at all exposure levels exhibited a focal to multifocal infiltration of the nasal epithelium with neutrophils and eosinophils graded as trace to mild. A luminal exudate was present only on one and three males from the mid and high exposure groups, respectively. These changes in the nasal passages were indicative of irritation and judged to be reversible.
The only other histopathological change that was statistically significant (p < 0.05) relative to control animals was an increased amount of hemosiderin pigment in the red pulp from spleens of female rats in the high-exposure group.

Dose descriptor:

NOAEC

Remarks:

(systemic)

Effect level:

3.3 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: temporarily reduced body weight in both sexes, not considered as adverse effect

Dose descriptor:

LOAEC

Remarks:

(systemic)

Effect level:

9.8 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced body weight in both sexes, increased amount of hemosiderin pigment in the red pulp from spleens of female rats

Dose descriptor:

LOAEC

Remarks:

(local)

Effect level:

1.1 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: nasal and ocular irritations, discharges, hyperplastic and metaplastic changes in the nasal tissue, focal to-multiofocal infiltration of the nasal epithelium

Critical effects observed:

not specified

Table 1: Body weights of male and female rats exposed to atmospheres containing maleic anhydride

Total maleic (mg/m³)
	0		1.1		3.3		9.8
Month	M	F	M	F	M	F	M	F
0	268a	188	269	187	267	187	269	185
1	385	252	374	250	372	247	371	251
2	423	279	416	275	400	265	395b	263b
3	467	300	446	293	437b	285	417b	276b
4	480	312	471	303	456	293	430b	286b
5	500	326	495	318	480	302b	448b	294b
6	526	342	524	332	510	320	482b	320

a Mean gram/rat for rats/group

b Significantly different from control.

Table 2: Histopathological observations in nasal tissue from rats exposed to atmospheres containing maleic anhydride

Total maleic (mg/m³)
Histopathological observation	0	1.1	3.3	9.8
Mucosa epithelial hyperplasia focal/multifocal, septum/ turbinates
M	0/0a	13/40	7/93	0/80
F	0/0	40/33	27/67	0/93
Mucosa, squamous metaplasia focal/ multifocal, septum/turbinates
M	0b	13	13	73
F	0	0	13	87

a Perrcentage of animals with trace grade/percentage of animals with mild grade . Tissues from 15 rats/sex/group were examined .

b Percentage of animals with observation . Tissues from 15 rats/sex/group were examined.

Conclusions:

In a 6-month inhalation study performed with rats, test item related effects on body weight and local effects in the respiratory tract were observed. Based on the results, the NOAEC (systemic) is considered to be 3.3 mg/m³ air and the LOAEC (local) 1.1 mg/m³ air.

Executive summary:

In a 6 -month inhalation toxicity study maleic anhydride was administered to 15 rats/sex/concentration by whole body exposure at concentrations of 0, 1.1, 3.3 or 9.8 mg/m³ air (analytical) for 6 hours per day, 5 days/week for a total of 6 months.

Nasal and ocular irritations were observed at all maleic anhydride-exposed animals during the study. High-exposure rats exhibited a red-tinged nasal discharge, isolated cases of ocular discharge and sneezing. These effects were less severe in animals from the low- and mid-exposure groups.Then, statistically significant reductions in body weights were observed in the male rats in the mid-exposure group on days 78, 92, 106, and 127; and in the high-exposure group on days 40 through 189 (p<0.05). Statistically significant reductions in body weights were observed in the female rats in the mid-exposure groups on days 54, 127, 141, 148, 155, an 156; and in the high-exposure group on days 37, 47, 57, 71, 85-176, and 189 [<10%, (p<0.05)]. However, at the end of the treatment period, terminal body weights were reduced only for male rats from the high-exposure group. There were no statistically significant effect on body weight at the low-exposure group for either sex of rat. Significant decrease in monocyte count in high dose male rats; however, no biological significance was attributed to these values since they were within the normal range af variation. Furthermore, clinical chemistry results indicated a few differences which were statistically different from the control values; however, none of these differences were considered to be treatment related or consistent in both sexes. Female rats in the low-dose group had a significantly reduced urine volume (- 59%) with an increased specific gravity (+ 3%). These observations were not dose related and thus not considered related to treatment.

Organ weight changes were observed in rats (8 of 9 tissues weighed). The only changes present in both the mid- and high-exposure groups were increased relative pituitary weight in female rats (+ 23%/+ 30%), increased relative adrenal weight (females: + 28%/36%; males: + 19% in  the high-exposure group) and reduced absolute (- 26%/- 28%) and relative thyroid weight (- 24%/- 20%) in male rats. Since the histopathology data did not provide evidence of tissue damage, these changes were not considered to be related to treatment.

Hyperplastic changes in the nasal tissues, which ranged in grade of severity from trace to mild, were present in all maleic anhydride exposed rats. Both the incidence and grade appeared to be exposure-related. Metaplastic changes in the nasal tissues were present in rats at all exposure levels and the incidence increased in a nonlinear fashion. Rats at all exposure levels exhibited a focal to multifocal infiltration of the nasal epithelium with neutrophils and eosinophils graded as trace to mild. A luminal exudate was present only on one and three males from the mid and high exposure groups, respectively. These changes in the nasal passages were indicative of irritation and judged to be reversible. The only other histopathological change that was statistically significant (p < 0.05) relative to control animals was an increased amount of hemosiderin pigment in the red pulp from spleens of female rats in the high-exposure group.

The NOAEC (systemic) is 3.3 mg/m³ based on the transient reduced body weight. The LOAEC (local) is 1.1 mg/m³ air based on the local effects in the respiratory tract observed at all applied concentration levels.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

3.3 mg/m³

Study duration:

subchronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Organ:

other: general toxicity (decreased body weights)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

Evaluation of maleic anhydride toxicity in rats, hamsters and monkeys after 6 months of inhalation administration.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Weight at study initiation: 230-310 g (males); 174-200 g (females)
- Age at study initiation: about 8 weeks
- Housing: single
- Diet: ad libitum (except during exposure)
- Water: ad libitum (except during exposure)
- Acclimation period: at least 2 weeks

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 15 m² cubical stainless-steel and glass chambers with pyramidal tops
- System of generating particulates/aerosols: Atmospheres containing the test material were generated by heating maleic anhydride, which has a melting point of 53°C, and by transporting vapors from the melt to the chambers with a stream of nitrogen gas. The overall quantity af maleic anhydride vaporized was determined by weighing the "boat" containing maleic anhydride prior to exposure and upon recrystallization of the compound after exposure.


TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations were sampled at least three times per day
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations in the chamber (total maleic; i .e ., maleic anhydride plus maleic acid) were monitored by drawing samples through Tenax columns and by quantifying the retained material, after thermal desorption into a nitrogen steam, using a gas chromatograph equipped with a flame ionization detector and a stainless-steel column packed with 1 .5% OV-l01 on 100-120 Chromosorb G-HP. The range of column and detector temperatures was 115 to 150°C and 205 to 220°C, respectively.

Duration of treatment / exposure:

6-months / 132 or 133 exposures

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

0 mg/m³ air (analytical)

Dose / conc.:

1.1 mg/m³ air (analytical)

Dose / conc.:

3.3 mg/m³ air (analytical)

Dose / conc.:

9.8 mg/m³ air (analytical)

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: 16- hour fasting period

Positive control:

no

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before and after each exposure

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week for the initial 2 months and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: monthly intervals
- Dose groups that were examined: on all test animals.

HAEMATOLOGY: Yes
- Animals fasted: Yes
- How many animals: 5 rats/sex/group from the control and high-exposure groups at 3 months and 5 rats/sex/group at 6 months
- Parameters checked: hemoglobin, hematocrit, total erythrocyte count, and total and differential leucocyts count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 3 and 6 months
- How many animals: 5 rats/sex/group from the control and high-exposure groups at 3 months and 5 rats/sex/group at 6 months
- Parameters checked: glucose, urea nitrogen, serum glutamic pyruvic transaminase activity, serum alkaline phosphatase activity, carbon dioxide, erythrocyte and plasma cholinesterase activity, and terminal brain cholinesterase activity

URINALYSIS: Yes
- Time schedule for collection of urine: 5 rats/sex/group from the control and high-exposure groups at 3 months and 5 rats/sex/group at 6 months
- Metabolism cages used for collection of urine: Yes
- Parameters checked: volume, pH, specific gravity, description of color and appearance, qualitative tests for albumin, glucose, bilirubin, ketones and occult blood and microscopic examination of the sediment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (on the control and high-exposure groups)

Statistics:

All statistical analyses compared the treatmant groups with the control group, by sex. Not all parameters were statistically evaluated at all intervals. Some parameters at some intervals were evaluated statistically after a review of the data indicated that a possible exposure related effec may have been present. The hematological, biochemical and urinalysis parameters (terminal) and absolute and relative organ weights (terminal) were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie using Dunnett's multiple comparison tables to judge significance of differences.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

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:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

For further details, see section below "Details on results".

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: Survival was >90% in all groups. Nasal and ocular irritations were observed at all maleic anhydride-exposed animals during the study. High-exposure rats exhibited a red-tinged nasal discharge, isolated cases of ocular discharge and sneezing. These effects were less severe in animals from the low- and mid-exposure groups.

BODY WEIGHT AND WEIGHT GAIN: Statistically significant reductions in body weights were observed in the male rats in the mid-exposure group on days 78, 92, 106, and 127; and in the high-exposure group on days 40 through 189 (p<0.05). Statistically significant reductions in body weights were observed in the female rats in the mid-exposure groups on days 54, 127, 141, 148, 155, an 156; and in the high-exposure group on days 37, 47, 57, 71, 85-176, and 189 [<10%, (p<0.05)]. However, at the end of the treatment period, terminal body weights were reduced only for male rats from the high-exposure group. There were no statistically significant effect on body weight at the low-exposure group for either sex of rat.

HAEMATOLOGY: significant decrease in monocyte count in high dose male rats; no biological significance was attributed to these values since they were within the normal range af variation

CLINICAL CHEMISTRY: Clinical chemistry results indicated a few differences which were statistically different from the control values; however, none of these differences were considered to be treatment related or consistent in both sexes. These differences were:
- glucose was reduced in males (- 9%) and increased in females (+ 17%) of the mid dose group after 6 months while both sexes of the high dose group had reduced values after 3 months (-18% and -21%, respectively);
- serum CO2 levels were slightly elevated in the high dose males after 6 months (+ 11%);
- urea nitrogen was slighly increased in high dose females after 6 months (+ 26%);
- red blood cell cholinesterase activity was reduced in mid dose males at 6 months (-17%) and brain cholinesterase was increased in mid (+ 27%) and high dose males (+ 21%) at 6 months

URINALYSIS: Female rats in the low-dose group had a significantly reduced urine volume (- 59%) with an increased specific gravity (+ 3%). These observations were not dose related and thus not considered related to treatment.

ORGAN WEIGHTS: Organ weight changes were observed in rats (8 of 9 tissues weighed). The only changes present in both the mid- and high-exposure groups were increased relative pituitary weight in female rats (+ 23%/+ 30%), increased relative adrenal weight (females: + 28%/36%; males: + 19% in the high-exposure group) and reduced absolute (- 26%/- 28%) and relative thyroid weight (- 24%/- 20%) in male rats. Since the histopathology data did not provide evidence of tissue damage, these changes were not considered to be related to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC: Hyperplastic changes in the nasal tissues, which ranged in grade of severity from trace to mild, were present in all maleic anhydride exposed rats. Both the incidence and grade appeared to be exposure-related. Metaplastic changes in the nasal tissues were present in rats at all exposure levels and the incidence increased in a nonlinear fashion. Rats at all exposure levels exhibited a focal to multifocal infiltration of the nasal epithelium with neutrophils and eosinophils graded as trace to mild. A luminal exudate was present only on one and three males from the mid and high exposure groups, respectively. These changes in the nasal passages were indicative of irritation and judged to be reversible.
The only other histopathological change that was statistically significant (p < 0.05) relative to control animals was an increased amount of hemosiderin pigment in the red pulp from spleens of female rats in the high-exposure group.

Dose descriptor:

NOAEC

Remarks:

(systemic)

Effect level:

3.3 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: temporarily reduced body weight in both sexes, not considered as adverse effect

Dose descriptor:

LOAEC

Remarks:

(systemic)

Effect level:

9.8 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced body weight in both sexes, increased amount of hemosiderin pigment in the red pulp from spleens of female rats

Dose descriptor:

LOAEC

Remarks:

(local)

Effect level:

1.1 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: nasal and ocular irritations, discharges, hyperplastic and metaplastic changes in the nasal tissue, focal to-multiofocal infiltration of the nasal epithelium

Critical effects observed:

not specified

Table 1: Body weights of male and female rats exposed to atmospheres containing maleic anhydride

Total maleic (mg/m³)
	0		1.1		3.3		9.8
Month	M	F	M	F	M	F	M	F
0	268a	188	269	187	267	187	269	185
1	385	252	374	250	372	247	371	251
2	423	279	416	275	400	265	395b	263b
3	467	300	446	293	437b	285	417b	276b
4	480	312	471	303	456	293	430b	286b
5	500	326	495	318	480	302b	448b	294b
6	526	342	524	332	510	320	482b	320

a Mean gram/rat for rats/group

b Significantly different from control.

Table 2: Histopathological observations in nasal tissue from rats exposed to atmospheres containing maleic anhydride

Total maleic (mg/m³)
Histopathological observation	0	1.1	3.3	9.8
Mucosa epithelial hyperplasia focal/multifocal, septum/ turbinates
M	0/0a	13/40	7/93	0/80
F	0/0	40/33	27/67	0/93
Mucosa, squamous metaplasia focal/ multifocal, septum/turbinates
M	0b	13	13	73
F	0	0	13	87

a Perrcentage of animals with trace grade/percentage of animals with mild grade . Tissues from 15 rats/sex/group were examined .

b Percentage of animals with observation . Tissues from 15 rats/sex/group were examined.

Conclusions:

In a 6-month inhalation study performed with rats, test item related effects on body weight and local effects in the respiratory tract were observed. Based on the results, the NOAEC (systemic) is considered to be 3.3 mg/m³ air and the LOAEC (local) 1.1 mg/m³ air.

Executive summary:

In a 6 -month inhalation toxicity study maleic anhydride was administered to 15 rats/sex/concentration by whole body exposure at concentrations of 0, 1.1, 3.3 or 9.8 mg/m³ air (analytical) for 6 hours per day, 5 days/week for a total of 6 months.

Nasal and ocular irritations were observed at all maleic anhydride-exposed animals during the study. High-exposure rats exhibited a red-tinged nasal discharge, isolated cases of ocular discharge and sneezing. These effects were less severe in animals from the low- and mid-exposure groups.Then, statistically significant reductions in body weights were observed in the male rats in the mid-exposure group on days 78, 92, 106, and 127; and in the high-exposure group on days 40 through 189 (p<0.05). Statistically significant reductions in body weights were observed in the female rats in the mid-exposure groups on days 54, 127, 141, 148, 155, an 156; and in the high-exposure group on days 37, 47, 57, 71, 85-176, and 189 [<10%, (p<0.05)]. However, at the end of the treatment period, terminal body weights were reduced only for male rats from the high-exposure group. There were no statistically significant effect on body weight at the low-exposure group for either sex of rat. Significant decrease in monocyte count in high dose male rats; however, no biological significance was attributed to these values since they were within the normal range af variation. Furthermore, clinical chemistry results indicated a few differences which were statistically different from the control values; however, none of these differences were considered to be treatment related or consistent in both sexes. Female rats in the low-dose group had a significantly reduced urine volume (- 59%) with an increased specific gravity (+ 3%). These observations were not dose related and thus not considered related to treatment.

Organ weight changes were observed in rats (8 of 9 tissues weighed). The only changes present in both the mid- and high-exposure groups were increased relative pituitary weight in female rats (+ 23%/+ 30%), increased relative adrenal weight (females: + 28%/36%; males: + 19% in  the high-exposure group) and reduced absolute (- 26%/- 28%) and relative thyroid weight (- 24%/- 20%) in male rats. Since the histopathology data did not provide evidence of tissue damage, these changes were not considered to be related to treatment.

Hyperplastic changes in the nasal tissues, which ranged in grade of severity from trace to mild, were present in all maleic anhydride exposed rats. Both the incidence and grade appeared to be exposure-related. Metaplastic changes in the nasal tissues were present in rats at all exposure levels and the incidence increased in a nonlinear fashion. Rats at all exposure levels exhibited a focal to multifocal infiltration of the nasal epithelium with neutrophils and eosinophils graded as trace to mild. A luminal exudate was present only on one and three males from the mid and high exposure groups, respectively. These changes in the nasal passages were indicative of irritation and judged to be reversible. The only other histopathological change that was statistically significant (p < 0.05) relative to control animals was an increased amount of hemosiderin pigment in the red pulp from spleens of female rats in the high-exposure group.

The NOAEC (systemic) is 3.3 mg/m³ based on the transient reduced body weight. The LOAEC (local) is 1.1 mg/m³ air based on the local effects in the respiratory tract observed at all applied concentration levels.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

1.1 mg/m³

Study duration:

subchronic

Species:

rat

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

Suitable data were available to assess the toxicity of maleic anhydride after repeated dose exposure. Therefore, the available data from repeated dose toxicity studies conducted with maleic anhydride were used in a weight of evidence approach to assess the specific target organ toxicity of the target substance.

A two years chronic oral study was conducted with male and female Fisher 344 rats which received 0, 10, 32, and 100 mg/kg bw/d maleic anhydride in the feed (126 animals per sex per dose; besides the females which got 10 mg/kg bw: 123 animals). Scheduled interim termination timepoints were at 6, 12, and 18 months with final study termination at 24 months. There was only marginal toxicity which was evidenced by small (< 6%) but dose-related, decrease in body weights of male rats fed 32 and 100 mg/kg bw/day compared to the controls. The female rats fed 32 and 100 mg/kg bw/day also had reduced body weights, but the reductions were smaller and of shorter duration than those observed in males. Food consumption was also slightly reduced during limited periods during the study for animals in the mid- and high-dose groups. Neither neurologic nor ophthalmologic evaluations revealed differences between treated and control animals. There was a high incidence of cataracts in the animals of this study, with 100% of the animals examined at 18 month and at study termination bearing cataracts. The severity of these cataracts was independent of maleic anhydride consumption. Haematology, clinical chemistry, gross or histopathological evaluations (which included the kidneys) showed no differences between treated and control animals that were not considered related to maleic anhydride exposure. The NOEL (males and females) was considered to be 10 mg/kg bw/day. Due to the small but dose related reduced body weights the LOEL (males and females) was assessed as 32 mg/kg bw/day.

Then, a 90 days feeding study divided in two phases with male and female Sprague-Dawley rats (15 animals per sex per dose) which received during the first phase 0, 100, 250 or 600 mg/kg bw/day maleic anhydride and the second phase 0, 20 or 40 mg/kg bw/day maleic anhydride was conducted. At 600 mg/kg bw/day, there was slight proteinuria in both sexes, increased relative liver weight in males, increased relative/absolute kidney weights in both sexes (p < 0.05). At 250 mg/kg bw/day, there were increased relative/absolute kidney weights in males (p < 0.05). Grossly observed kidney changes were seen in males fed maleic anhydride at 100, 250, and 600 mg/kg bw/day. The changes were characterized by increased size, pale discoloration, and evidence of dilated tubules in the cortex. Microscopically, the kidneys showed varying degrees of nephrosis, being most severe in the high-dose group. These changes consisted of diffuse tubular dilatation, hypertrophy, and degeneration and regeneration of the tubular cells in the cortical portion of the nephron. A decrease in severity of these changes was observed at lower dose levels, with the kidneys from the 100 mg/kg dose group showing minimal laterations. Similar changes were observed in the kidneys of females, but were generally limited to the high-dose group and were much less severe. In the lung of one male at both 250 and 600 mg/kg/day, there was a singular nodular lesion observed grossly that was microscopically diagnosed as a pulmonary adenoma. Therefore, for the first phase the NOAEL (female) was 250 mg/kg bw/day and the NOAEL (male) was considered to be 100 mg/kg bw/day due to renal changes. For the second phase the NOEL (male/female) was considered to be 40 mg/kg bw/day, test item related effects were not observed among all rats tested.

In a 90 / 183 day study 50 male Sprague-Dawley rats per dose received 0, 250 and 600 mg/kg bw/day with the diet. No statistically significant changes in mean body weights and food consumption between treated and control animals were reported. Besides, there were no meaningful (or significant) differences in haematology, urinalysis, urine concentration, or clinical chemistry tests. At the 90-day timepoint, the relative liver weight for the rats in the high-dose level and the absolute/relative kidney weights for both the 250 and 600 mg/kg groups were significantly higher compared to the controls (p< 0.05).Treatment-related changes were present in the kidneys of rats terminated at 90 and 183 days. The observed changes indicated a marked accentuation of the spontaneously occurring findings seen in the control animals. The changes in the controls and treated animals included: individual tubules that were dilated and contained eosinophilic staining casts, granular degeneration of the epithelial cells lining these tubules, tubular collapse and atrophy with peritubular fibrosis, focal mononuclear inflammatory cell infiltrates, glomeruli that showed thickening of the basement membrane, thickening and epithelialization of Bowmans Capsule, and occasionally showed either focal or diffuse sclerosis of the glomerular tufts. Both tubular and glomerular changes in treated animals were more severe than controls, and much more severe in the treated rats at 183 days compared to 90 days. In addition to the focal nature of the lesion (in controls), the tubules throughout the cortex of treated rats showed a generalized dilatation and hypertrophy. There were more degenerative tubules and tubules showing mitotic activity in treated versus controls. The degree of degenerative, hypertrophic, and regenerative changes was dose-related. In many of the 600 mg/kg dose group animals, there was a marked decrease in the amount of functional tissue in the kidney. Livers of maleic anhydride-treated rats at 183 days showed changes characterized by swollen individual hepatocytes having vacuolated cytoplasm. Due to changes in the kidney/kidney weight the LOAEL (male) was considered to be 250 mg/kg bw/day.

Also, a 90 day feeding study (0, 20, 40, and 60 mg/kg bw/day) with 4 Beagle dogs per sex per dose was conducted. Body weights and food consumption were recorded, and the dogs were observed for signs of toxicity. Clinical parameters were evaluated by determination made prior to the beginning of the study and at intervals throughout the test period. These included haematological studies, urinalysis and various clinical chemistry studies. Gross examination was conducted at necropsy after the exposure period, organ weights were examined, and tissues were obtained for histopathological evaluation. Food intake was decreased for the first few weeks in both sexes of dogs receiving 60 mg/kg bw/day. However, these changes were transient and did not result in longterm body weight reduction. Haematological studies conducted after 83 days of exposure indicated equivocal decreases in packed cell volume and hemoglobin concentration of male dogs receiving 60 mg/kg bw/day. This effect was not considered to be "adverse". No other changes were observed that were considered relatable to the ingestion of diets containing maleic anhydride. Therefore the NOAEL of the study is 60 mg/kg bw/day.

 

In a subchronic inhalation studies 15 male/female CD rats, 15 male/female Engle hamsters or 3 Rhesus monkeys per sex per dose were exposed by inhalation (whole body) to 0, 1.1, 3.3 and 9.8 mg/m³(0, 0.3, 0.8, and 2.4 ppm) maleic anhydride for six hours/day, five days/week for six months. Body weights were decreased in male and female rats in the mid- and high-exposure groups at intervals during the study. However, at study termination, body weights were decreased only in the male animals of the high-exposure group (6 - 8%). All other effects were limited to the respiratory tract and eye. In the high-exposure groups, nasal discharge, ocular irritation, and slight dyspnea with coughing and sneezing were observed. The effects were less severe in the animals from the mid- and low-exposure groups. In the rats and hamsters (but not monkeys), hyperplastic changes in the nasal tissues were present in the mid- and high-exposure groups only. Also in the rats and hamsters, metaplastic changes in the nasal tissues were present in all exposure groups. In all species, there was some mucosal and/or submucosal infiltration of neutrophils into the nasal tissues at all exposure groups. All of these effects were considered indicative of irritation and judged to be reversible. The NOAEC (systemic) for male and female rats is considered to be 3.3 mg/m³ (the temporarily reduced body weight in both sexes was not regarded as an adverse effect). The LOAEC (systemic) of 9.8 mg/m³for male and female rats is based on decreased body weights for longer time periods in both sexes and increased amounts of hemosiderin pigment in the red pulp from spleens of female rats. As there were no adverse effects detected in hamsters and monkeys the NOEC (systemic) for hamsters and monkeys (males and females) is assessed as 9.8 mg/m³. Based on localized nasal irritation effects in all tested animal species (males and females), the LOAEC(local) is considered as 1.1 mg/m³.

A subacute inhalation study (0, 0.01, 0.03, 0.1 mg/L maleic anhydride) with male and female Sprague-Dawley rats (10 per sex per dose) was also conducted. Evidence of nasal and ocular irritation (concentration-dependent) occurred at all treatment levels after 1 month. At the highest concentration, ocular and nasal discharge, periodic nasal bleeding and respiratory distress were seen. Less severe effects occurred at lower concentrations. Reduced body weight gain (both sexes) and food consumption (females only) occurred at 32 and 86 mg/m³. There were no haematological, clinical chemistry or urine changes that could be conclusively attributed to treatment. A number of organ weight differences were seen but these were considered to be related to body weight differences. There was an increased incidence of hemorrhagic lung foci in the mid- and high-exposure groups compared to controls. There were also treatment-related effects (concentration-related) in the nasal turbinates, trachea and lungs. These included epithelial hyperplasia and presence of inflammatory exudate in the nasal turbinates and trachea, and in the lungs, epithelial hyperplasia, squamous metaplasia and intra-alveolar hemorrhage. The severity of the microscopic changes was generally described as slight to moderate. Also, keratitis or corneal vascularization was noted in the eyes of a number of animals from the high-dose group, which was considered by the authors to be a local effect and not a reflection of systemic toxicity. Due to changes in the organ weights/reduced body weight, the LOAEC (systemic) was considered to be 0.01 mg/L air. There were also local effects in the respiratory tract at all applied dose levels and therefore a LOAEC (local) was also defined as 0.01 mg/L air.

Since rodents (rats & hamsters) are obligatory nasal breathers, it is difficult to extrapolate the significance of these findings to humans or other species which are capable of breathing through their mouths.

Justification for classification or non-classification

Based on the available data and in accordance with ATP13 to CLP Regulation 1272/2008 classification as STOT RE 1, H372 (causes damage to organs < Respiratory system> through prolonged or repeated exposure <by inhalation>) is warranted.