N,N-dimethylbutylamine

Endpoint summary

Description of key information

No data were located for N,N-dimethylbutylamine (DMBA). Therefore, a read across approach from data on n-butanol, n-butylacetate and dimethylamine was undertaken.

It is considered justified to utilise information on dimethylamine in a read across approach since, like the registered substance, it is an aliphatic amine with saturated alkyl groups. Furthermore, dimethylamine is considered to be a potential metabolite of the registered substance.

It is considered justified to utilise information on n-butanol in a read across approach since it is a potential metabolite of the registered substance.

It is considered justified to utilise information on n-butylacetate in a read across approach since its metabolite (n-butanol) is also a potential metabolite of the registered substance.

ORAL

- Sub-Chronic, n-butanol (K1)

The No Observed Adverse Effect Level was determined to be 125 mg/kg bw/day (US EPA, 1986). This value is taken forward for risk assessment.

INHALATION

- Chronic, dimethylamine (K2)

In this study (Buckley et al., 1985) the LOAEC for local effects was 10 ppm dimethylamine (18.7 mg/m³), as evidenced by lesions of the respiratory and olfactory epithelium of rats and mice. The NOAEC for systemic toxicity was 50 ppm DMA (93.5 mg/m³) in both species. Local irritation is the primary mode of action of all free aliphatic amines, and this finding can be transferred to N,N-dimethylbutylamine for assessment.

A follow up study (Gross, 1987) confirmed the previously reported respiratory metaplasia seen in the olfactory region of the nose following chronic exposure of rats to DMA (Buckley et al., 1985).

- Sub-Chronic, n-butyl acetate (K1)

Under the conditions of the study investigating neurotoxicity (David, 1998) the No Observed Adverse Effect Concentration was determined to be 500 ppm (ca. 2350 mg/m³).

In a repeated dose toxicity test (David, 2001), the No Observed Adverse Effect Concentration was determined to be 500 ppm (ca. 2350 mg/m³) based on reduced body weight and feed consumption noted for the 1500 and 3000 ppm groups. However, there was no systemic or organ-specific toxicity. Degeneration of the olfactory epithelium at concentrations of 1500 and 3000 ppm was observed in areas of the nasal cavity that have demonstrated carboxylesterase activity (Bogdanffy, 1990: Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach. Environmental Health Perspectives 85, 177–186), but there was no evidence of pulmonary toxicity.

The Buckley data are taken forward for risk assessment, as these studies provide a more conservative effect level.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

other: read-across target

Principles of method if other than guideline:

Four groups of male and female rats (30/sex/group) were administered the test material daily by gavage at 0, 30, 125 or 500 mg/kg bw/d for either 6 or 13 weeks.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan, U.S.A.
- Age at study initiation: 36-37 d
- Mean weight at study initiation: males 90 g, females 86 g
- Housing: individually
- Diet (e.g. ad libitum): Purina Certified Rodent Laboratory Chow #5002 (pellet)
- Water (e.g. ad libitum): filtered municipal water
- Acclimation period: 7 days before the pre-treatment week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Humidity (%): 48 ± 9
- Photoperiod (hrs dark / hrs light): 12:12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions of butanol in deionised water were used.

VEHICLE
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 10 mL/kg was the constant dosing volume

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

GC-FI

Duration of treatment / exposure:

6 (interim sacrifice) or 13 weeks

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

125 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 (further 10 were sacrificed prior to dosing for determination of clinicopathological baseline levels)

Control animals:

yes, concurrent vehicle

Positive control:

no data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly

FOOD CONSUMPTION: Yes
-Time schedule: Food consumption was recorded weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmic examination was conducted prior to treatment and during week 13 before final necropsy.
- Dose groups that were examined: no data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the start of the study, during week 6 and during week 13
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 males and 10 females
- parameters: haemoglobin (HGB), haematocrit (PCV), erythrocyte count (RBC), mean cell volume (MCV), mean cell haemoglobin (MCH),mean cell haemoglobin concentration (MCHC) total and differential leucocyte counts (WBC), estimated platelet count (PLT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before the start of the study, during week 6 and during week 13
- Animals fasted: No data
- How many animals: 10 males and 10 females
- parameters: alkaline phosphatase (Alk phos) blood urea nitrogen (BUN), glutamate pyruvate transaminase (SGPT), glutamate oxaloacetate transaminase (SGOT), glucose (Gluc), total protein (TP), albumin (Alb), A/G ratio (calculated), globulin (calculated), total bilirubin (Tot. bili.), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphate (Phos), carbon dioxide (TCO2), total serum cholesterol (Chol), creatinine.

URINALYSIS: Yes
- Time schedule for collection of urine: before the start of the study, during week 6 and during week 13
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- parameters: pH, specific gravity, glucose, protein, ketones, bilirubin, urobilinogen, microscopy of sediment

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: Ten male and ten female rats from each group were necropsied on study days 43 to 44 and the remaining animals on study days 92 to 93. Gross pathology of all animals was assessed and organs from animals necropsied on study days 92 to 93 were weighed.

HISTOPATHOLOGY: Yes: A complete histopathological investigation was made of all animals of the control and high-dose groups. In the low and mid-dose groups, histopathology included the liver, kidney, and heart from all animals and all gross lesions. All animals found dead or killed in extremis were also microscopically examined.

Statistics:

no data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ataxia and hypoactivity (lasting less than 1 h) were observed 2 to 3 minutes after dosing in both sexes of the high-dose group (500 mg/kg bw/d) during the final 6 weeks of dosing. Such ataxia and hypoactivity are typically seen following high oral doses of alcohols. The rapid induction/remission of these effects and the reported increased incidence after the interim kill may be due to the fact that personnel were able to collect post-dose observations more quickly since fewer animals required dosing.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No significant changes between treated groups and controls were observed concerning mortality (three rats were found dead or sacrificed in extremis, but these deaths could not be attributed to the test article.).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

no significant changes between treated groups and controls were observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

no significant changes between treated groups and controls were observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

no significant changes between treated groups and controls were observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the interim clinical pathological evaluation, red blood cell count (RBC), packed cell volume (PCV), and haemoglobin (HGB) averages of the 500 mg/kg/day dose group females were 5 % below control averages. Although these differences were statistically significant, they were small and no differences between the parameters were observed in the males of the interim evaluation or between control and treated groups of either sex at the final evaluation. Therefore, even if the lower red blood cell parameters in the 500 mg/kg/day females were an actual treatment-related effect, it was small and transitory and thus not considered as adverse.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

no significant changes between treated groups and controls were observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

no significant changes between treated groups and controls were observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

no significant changes between treated groups and controls were observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

no significant changes between treated groups and controls were observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

no significant changes between treated groups and controls were observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs of CNS depression (ataxia and hypoactivity)

Critical effects observed:

not specified

Conclusions:

The No Observed Adverse Effect Level was determined to be 125 mg/kg bw/day.

Executive summary:

Four groups of male and female rats were administered the test material daily by gavage at 0, 30, 125 or 500 mg/kg bw/d for either 6 or 13 weeks. Dosing solutions of butanol in deionised water were used.

30 rats per sex per group were dosed, with a further 10 being sacrificed prior to dosing for determination of clinicopathological baseline levels. Ten male and ten female rats from each group were necropsied on study days 43 to 44 (interim sacrifice) and the remaining animals on study days 92 to 93.

Ataxia and hypoactivity (lasting less than 1 h) were observed 2 to 3 minutes after dosing in both sexes of the high-dose group (500 mg/kg bw/d) during the final 6 weeks of dosing. Such ataxia and hypoactivity are typically seen following high oral doses of alcohols. The rapid induction/remission of these effects and the reported increased incidence after the interim kill may be due to the fact that personnel were able to collect post-dose observations more quickly since fewer animals required dosing.

At the interim clinical pathological evaluation, red blood cell count (RBC), packed cell volume (PCV), and haemoglobin (HGB) averages of the 500 mg/kg/day dose group females were 5 % below control averages. Although these differences were statistically significant, they were small and no differences between the parameters were observed in the males of the interim evaluation or between control and treated groups of either sex at the final evaluation. Therefore, even if the lower red blood cell parameters in the 500 mg/kg/day females were an actual treatment-related effect, it was small and transitory and thus not considered as adverse.

Under the conditions of the study the No Observed Adverse Effect Level was determined to be 125 mg/kg bw/day.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

It is considered justified to utilise information on this substance in a read across approach since it is a potential metabolite of the registered substance.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs of CNS depression (ataxia and hypoactivity)

Critical effects observed:

not specified

Conclusions:

The No Observed Adverse Effect Level was determined to be 125 mg/kg bw/day.

Executive summary:

It is considered justified to utilise information on n-butanol in a read across approach since it is a potential metabolite of the registered substance.

Four groups of male and female rats were administered the test material daily by gavage at 0, 30, 125 or 500 mg/kg bw/d for either 6 or 13 weeks. Dosing solutions of butanol in deionised water were used.

30 rats per sex per group were dosed, with a further 10 being sacrificed prior to dosing for determination of clinicopathological baseline levels. Ten male and ten female rats from each group were necropsied on study days 43 to 44 (interim sacrifice) and the remaining animals on study days 92 to 93.

Ataxia and hypoactivity (lasting less than 1 h) were observed 2 to 3 minutes after dosing in both sexes of the high-dose group (500 mg/kg bw/d) during the final 6 weeks of dosing. Such ataxia and hypoactivity are typically seen following high oral doses of alcohols. The rapid induction/remission of these effects and the reported increased incidence after the interim kill may be due to the fact that personnel were able to collect post-dose observations more quickly since fewer animals required dosing.

At the interim clinical pathological evaluation, red blood cell count (RBC), packed cell volume (PCV), and haemoglobin (HGB) averages of the 500 mg/kg/day dose group females were 5 % below control averages. Although these differences were statistically significant, they were small and no differences between the parameters were observed in the males of the interim evaluation or between control and treated groups of either sex at the final evaluation. Therefore, even if the lower red blood cell parameters in the 500 mg/kg/day females were an actual treatment-related effect, it was small and transitory and thus not considered as adverse.

Under the conditions of the study the No Observed Adverse Effect Level was determined to be 125 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subchronic

Species:

rat

System:

central nervous system

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

other: read-across target

Principles of method if other than guideline:

Male and female F-344 rats and B6C3F1 mice were exposed by inhalation to 0, 10, 50, or 175 ppm dimethylamine (DMA) for 6 hr/day, 5 days/week for 12 months. Groups of 9-10 male and female rats and mice were necropsied after 6 and 12 months of exposure.
The purpose of this study was to investigate the toxicity associated with chronic inhalation exposure of F-344 rats and B6C3F1 mice to DMA for 2 years. This report summarises the clinical and pathologic data found for the first 12-month period.

GLP compliance:

not specified

Limit test:

no

Species:

other: rat and mouse

Strain:

other: F-344 rats and B6C3F1 mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River breeding laboratories, Kingston, New York, USA
- Age at study initiation: 4-8 weeks
- Housing: individually in hanging stainless steel wire mesh cages in the exposure chambers
- Diet: NIH-07 open formula diet, Ziegler Brothers, Gardners, Pa.; analyzed for contaminants by Lancaster Labs, Lancaster, Pa., ad libitum during periods of non-exposure
- Water: tap water via an automatic watering system, ad libitum during periods of non-exposure
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 - 76 °F
- Humidity (%): 45 - 65 % ( real: 35 - 74 %)
- Air: airflow of 2200 liters/min
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: n.a.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: test atmospheres were generated by metering pure DMA directly from the cylinder via flowmeters (Fischer-Porter, Warminster, Pa., or Calibrating and Measuring Equipment, Inc., Manassas, Va.) into the supply air stream.
- Method of holding animals in test chamber: rats and mice were exposed and housed in 8-m3 stainless steel and glass whole body chambers operated with a dynamic airflow of approximately 2200 liters/min (HEPA-filtered room air) and at slightly subatmospheric pressure (0.2-0.3 in. of water). The cages within a rack were rotated once per week using a computer-generated randomisation procedure designed to ensure that each animal spent an equal amount of time in all areas of the chamber.

- Method of conditioning air: HEPA filter
- System of generating particulates/aerosols: DMA directly entered the air stream
- Temperature, humidity, pressure in air chamber: slightly subatmospheric pressure (0.2-0.3 in. of water).
- Air flow rate: 2200 liters/min
- Air change rate: approx. 15
- Method of particle size determination: n.a.

TEST ATMOSPHERE
- Brief description of analytical method used: IR spectrophotometer
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the test atmospheres was performed four times per hour by infrared spectrometry at a wavelength of 3.5 µm and a path length of 20.25 m (MIRAN 801, Foxboro-W&s, Norwalk, Conn.). The spectrophotometer was set to zero optical density with ultra zero air containing approximately 350 ppm Co, (Ma&son Gas, Morrow, Ga.) and adjusted to approximately 50 % relative humidity. Temperature and relative humidity were recorded hourly.

The mean time-weighted average (TWA) analytical chamber concentrations for the 12 month period, derived from daily TWA concentrations, with standard deviations and ranges were
175 ppm +/- 2.0 (167-188),
50.0 ppm +/- 1.0 (45.8-54.0), and
10.0 ppm +/- 0.3 (9.0-10.8).

The average percentage ratios of analytical/nominal concentrations (+/- standard deviations) were 81 +/- 8, 87 +/- 7, and 76 +/- 15 % for the 175-, 50-, and 10-ppm chambers, respectively, thus indicating a fairly constant loss of DMA on the chamber surfaces and animals for each chamber. The distribution of the analytical concentration of DMA in the chambers was determined to be uniform within +6 % of target concentrations.

Duration of treatment / exposure:

12 month

Frequency of treatment:

5 days/week, 6 hours/day

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

10 ppm (nominal)

Dose / conc.:

50 ppm (nominal)

Dose / conc.:

175 ppm (nominal)

No. of animals per sex per dose:

95 animals

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: dose levels were selected on the basis of preceding studies. F-344 rats exposed to 175 or 250 ppm DMA (6 hr/day, 5 days) or 500 ppm DMA (6 hr/day, 3 days) had ulcerative rhinitis, severe congestion, and squamous metaplasia in the respiratory tract.These lesions were most severe in the anterior sections of the nasal passages.

Observations and examinations performed and frequency:

The animals were observed for clinical abnormalities twice daily and were weighed once per week for the first 13 weeks, and biweekly thereafter.

Sacrifice and pathology:

After 6 and 12 months, 9-10 animals of each species, sex, and treatment group were fasted overnight and weighed just prior to necropsy. Male mice were not included in the 12-month sacrifice because of the high rate of unscheduled mortality in this group.

Animals were anesthetised with pentobarbital by i.p. injection, and blood was drawn from the heart for haematology and serum chemistry. Each animal was examined for gross abnormalities, and 45 tissues and any gross lesions were collected and placed in 10 % buffered formalin. The nasal passages were flushed, the lungs inflated, and the lumen of the gastrointestinal tract infused with formalin. The liver, kidneys, and brain were weighed. All tissues from the control and 175 ppm exposed animals and target tissues (nasal turbinates) from the 10- and 50- ppm exposed animals were processed for light microscopic examination. Tissues containing bone were placed in 10 % buffered formalin for 48 hr, desiccated, then rinsed with tap water for at least 4 hr, and replaced in formalin. Tissues were then dehydrated in graded alcohols, cleared with xylene, and embedded in paraffin wax. Transverse blocks of the nose were cut and prepared to yield histological sections at the following levels: (1) just anterior to the incisor teeth, (2) approximately one third of the distance from the posterior aspect of the incisor teeth to the incisive papilla, (3) at the incisive papilla, (4) at the crest of the second palatial ridge, and (5) at the centre of the second molar tooth. Embedded tissues were sectioned at 5 pm and stained with haematoxylin and eosin for light microscopic examination. For photography, selected tissues were removed from paraffin re-embedded in glycol methacrylate (GMA), and 2- to 3- am-thick sections were cut and stained with Lee’s methylene blue. Selected nasal sections were stained with Alcian blue at pH 2.5 for acidic mucous glycoproteins.

Other examinations:

Hematological data were collected using a Coulter S Plus II counter. Sodium and potassium determinations were performed with a flame photometer. Chloride analyses were performed with a Coming Chloride 920 M meter (Coming Scientific Inst., Mechield, Mass.). An Abbott VP clinical analyser was used for determination of serum chemistries.

Statistics:

Data for body weights, serum chemistry, haematology, and organ weights were analyzed using an analysis of variance. Dunnett’s test was employed to detect differences between control and treatment groups (Steel and Torrie, 1980). Red blood cell morphology and histopathological findings were analyzed using the Kolmorgomov-Smimov test (Daniel, 1978). In all cases, the preselected significance level was p ≤ 0.05.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

significant loss at 175 ppm ( 90 % of control)

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

decreased platelet counts (175 ppm), increased counts of atypical lymphocytes in females (175 ppm), decreased mean red blood cell volume (females, 175 ppm)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

decreased protein (175 ppm), increased alkaline phosphatase (females, 175 ppm)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

(1) LESIONS IN RESPIRATORY EPITHELIUM
The lesions in the respiratory area were most severe on the anterior septum, just posterior to the vestibule, and on the free margins of the naso- and maxilloturbinates, with lesser involvement of the lateral wall. In the 175 ppm exposure group, there was variable destruction of the anterior portions of the naso- and maxilloturbinates, and fenestration of the nasal septum. In areas of destruction of the turbinates and septum, the surface was covered by nonkeratinizing squamous epithelium. Evidence of both acute and chronic inflammatory response included focal to diffuse mucosal and submucosal infiltration of mononuclear leukocytes and some neutrophils. Exudate was minimal or absent. Other lesions included epithelial hypertrophy and hyperplasia, focal epithelial ulceration, and focal to diffuse squamous metaplasia. Goblet cell hyperplasia of mild to moderate severity was observed on the ventral aspect of the nasal septum in rats, but not in mice. Globules of eosinophilic material were observed in the respiratory epithelium of the anterior nasal passages. The globules were present in the basal half of the affected cells, and the dense, oval to round nucleus appeared to be compressed between the globules and the basement membrane. The cell type containing the globules was not identified. Small, basophilic bodies, which were laminated and presumably mineralized, with the globules in mice.
At the 50-ppm exposure level, lesions in the respiratory epithelium were minimal in both rats and mice. Changes were confined to focal squamous metaplasia on the free margins of the turbinates in mice after 6 months of exposure, and eosinophilic globules with mild inflammation and epithelial hypertrophy and hyperplasia after 12 months. A slightly higher incidence of chronic inflammation was observed in the vestibule and the respiratory epithelium of rats in the 10-ppm exposure group.

(2) LESIONS IN THE OLFACTORY REGION
DMA exposure induced a concentration-dependent destruction of the olfactory epithelium which was most severe in the middle third of the dorsal meatus, with variable involvement of the free margins of the ectoturbinates. The most widespread change, which was found consistently in DMA exposed rats and mice, was degeneration of olfactory sensory cells with variable vacuolation of the olfactory epithelium. Another lesion often observed in areas of the olfactory epithelium was characterized by accumulation of hyaline, eosinophilic material in sustentacular cells, which were often markedly hypertrophic. The eosinophilic material was also present as large globules in the overlying airway, suggesting that it may be a secretory product of the sustentacular cells. Similar material was present in the large submucosal glands at the junction of olfactory and respiratory epithelium. These lesions were almost always accompanied by atrophy of the olfactory nerves in the lamina propria. Bowman’s glands exhibited or focal hyperplasia. In the more severely affected cases, olfactory epithelium was replaced by well-differentiated, ciliated respiratory epithelium. These metaplastic areas of ciliated respiratory epithelium were found, in several cases, to be continuous with ciliated ducts of hypertrophic or hyperplastic Bowman’s glands. There were foci of fusiform cells near the basal layer in rats, but not in mice, in areas of respiratory metaplasia in the dorsal meatus. In the underlying, oedematous connective tissue, the basement membrane appeared thickened and separated from the epithelium. Basal cell hyperplasia was frequently observed in the olfactory epithelium of rats, but was not observed in mice. This lesion was characterized by a zone of polyhedral to fusiform cells lying beneath any remaining sustentacular cells. The basal cells had indistinct cytoplasmic boundaries and dark round nuclei and formed cords, sheets, or acinar arrangements. At the 50-ppm exposure level, lesions were much less severe than at 175 ppm and were confined to loss of sensory cells and olfactory nerves, primarily in the middle third of the dorsal meatus. However, most of the animals exposed to 50 ppm exhibited olfactory epithelial lesions. After 12 months, only a few animals at the l0-ppm exposure level were affected, and lesions were confined to focal degeneration of the olfactory epithelium in the dorsal meatus.

Details on results:

Treatment-induced lesions were confined to the nasal passages, and were very similar in nature and grade between the species and sexes. In mice, there was no apparent progression or increase in severity of the nasal lesions from 6 to 12 months, while in rats, increased exposure time was associated with more extensive involvement of the olfactory area. The lesions were present in two areas of the nose: (1) the respiratory epithelium and underlying tissues adjacent to the vestibule, and (2) the olfactory epithelium in the medial portion of the dorsal meatus with variable involvement of more posterior olfactory areas. There was a distinct concentration-response relationship for the severity and frequency of both lesions. After 12 months of treatment, clear effects were seen at 50 and 175 ppm, whereas at 10 ppm lesions were only seen in few animals.

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Deaths at 175 ppm (= 327 mg/m³) in rats and mice were secondary to severe local effects, but should nevertheless be considered to represent systemic toxicity.

Dose descriptor:

LOAEC

Remarks:

local irritation

Effect level:

10 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects on the respiratory epithelium in some of the rats and mice at 12 months after study initiation. Using a conversion factor (1 ppm = 1.87 mg/m³), 10 ppm equals 18.7 mg/m³

Critical effects observed:

not specified

Lesions in Nasal Passages of Rats and Mice Exposed to DMA for 12 Months
DMA (ppm)		Squamous epithelium (level I)^a	Respiratory epithelium (levels I, II)^b	Olfactory epithelium (levels II, III, IV)^c	Chronic inflammation (levels I, II, III)
0	Rats	—	—	+/-	+
	Mice	—	—	—	—
10	Rats	—	+/-	+	+
	Mice	—	+/-	+	—
50	Rats	—	+	++	++
	Mice	—	+	++	+
175	Rats	+/-	+++	+++	++
	Mice	+/-	+++	+++	+
Notes. — =No abnormality detected. +/- = very slight changes of doubtful significance, + = minimal change, ++ = moderate change, +++ = severe change. ^aFor levels of the nose, see Methods. ^bLesion largely confined to the respiratory epithelium adjacent to the vestibule. ^cLesions especially severe in the dorsal meatus. ^dLesions were somewhat more extensive in rats than mice.

 

Conclusions:

In this study the LOAEC for local effects was 10 ppm dimethylamine (DMA), as evidenced by lesions of the respiratory and olfactory epithelium of rats and mice. The NOAEC for systemic toxicity was 50 ppm DMA in both species. Local irritation is the primary mode of action of all free aliphatic amines, and this finding can be transferred to N,N-dimethylbutylamine for assessment.

Executive summary:

Rats (95 Fischer 344/sex and dose) and mice were exposed to atmospheres containing dimethylamine at 0, 10, 50, 175 ppm (exposure duration 12 months, 5 days/week, 6 hours/day). The concentration was monitored using IR spectrophotometry. Groups of rats (and mice) were sacrificed at 6 and 12 months after study initiation. Examinations included clinical signs body weights, clinical chemistry, haematology, necropsy and histopathology.

After 12 months of exposure, the only effect noted was a dose-dependent irritation of the nasal respiratory and olfactory epithelium, both in rats and mice, without any gender or species related differences. At 10 ppm the effects were minimal but present in several animals. Deaths occurred at 175 ppm in high incidence in male mice (Buckley et al, 1985).

 

The results indicate that the mode of action of the free amine is local irritation/corrosion at the point of contact, followed by inflammatory processes with concomitant alterations of the blood cell counts. Systemic toxicity is a minor issue and there was no indication in this study for any kind of systemic toxicity or target organ other than the airways. This result is in line with observations made with other saturated aliphatic amines. Therefore, the result can be read across to related substances, including N,N-dimethylamine, and can be used for assessment.. In this study the LOAEC for local effects was 10 ppm DMA, the NOAEC for systemic toxicity was 50 ppm DMA.

Reference 2

Endpoint:

chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

It is considered justified to utilise information on this substance in a read across approach since, like the registered substance, it is an aliphatic amine with saturated alkyl groups. Furthermore, dimethylamine is considered to be a potential metabolite of the registered substance.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Deaths at 175 ppm (= 327 mg/m³) in rats and mice were secondary to severe local effects, but should nevertheless be considered to represent systemic toxicity.

Dose descriptor:

LOAEC

Remarks:

local irritation

Effect level:

10 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects on the respiratory epithelium in some of the rats and mice at 12 months after study initiation. Using a conversion factor (1 ppm = 1.87 mg/m³), 10 ppm equal 18.7 mg/m³

Critical effects observed:

not specified

Conclusions:

In this study the LOAEC for local effects was 10 ppm dimethylamine (DMA), as evidenced by lesions of the respiratory and olfactory epithelium of rats and mice. The NOAEC for systemic toxicity was 50 ppm DMA in both species. Local irritation is the primary mode of action of all free aliphatic amines, and this finding can be transferred to N,N-dimethylbutylamine for assessment.

Executive summary:

It is considered justified to utilise information on dimethylamine in a read across approach since, like the registered substance, it is an aliphatic amine with saturated alkyl groups. Furthermore, dimethylamine is considered to be a potential metabolite of the registered substance.

Rats (95 Fischer 344/sex and dose) and mice were exposed to atmospheres containing dimethylamine at 0, 10, 50, 175 ppm (exposure duration 12 months, 5 days/week, 6 hours/day). The concentration was monitored using IR spectrophotometry. Groups of rats (and mice) were sacrificed at 6 and 12 months after study initiation. Examinations included clinical signs body weights, clinical chemistry, haematology, necropsy and histopathology.

After 12 months of exposure, the only effect noted was a dose-dependent irritation of the nasal respiratory and olfactory epithelium, both in rats and mice, without any gender or species related differences. At 10 ppm the effects were minimal but present in several animals. Deaths occurred at 175 ppm in high incidence in male mice (Buckley et al, 1985).

The results indicate that the mode of action of the free amine is local irritation/corrosion at the point of contact, followed by inflammatory processes with concomitant alterations of the blood cell counts. Systemic toxicity is a minor issue and there was no indication in this study for any kind of systemic toxicity or target organ other than the airways. This result is in line with observations made with other saturated aliphatic amines. Therefore, the result can be read across to related substances, including N,N-dimethylamine, and can be used for assessment.. In this study the LOAEC for local effects was 10 ppm DMA, the NOAEC for systemic toxicity was 50 ppm DMA.

Reference 3

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

other: read-across target

Principles of method if other than guideline:

Groups of six male Fischer-344 rats (CDF (F- 344)/CrlBR; Charles River Breeding Laboratories, Kingston, NY) were exposed to DMA at a concentration of 175 ppm, 6 hr per day for 1, 2, 4, or 9 days or 2 years.

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: 6 weeks
- Weight at study initiation: 241.67 ± 4.99,238.17 ± 4.29, 225.33 ± 1.84, 221.08 ± 3.17, and 346.50 ± 9.82 for animals exposed for 1, 2, 4, or 9 days or 2 years, respectively, and 228.25 ± 4.20 and 355.83 ± 11.92 for the acute and chronic control groups (values are expressed as means in grams ± SE).
- Housing: individually in hanging stainless steel wire-mesh cages in the exposure chambers, 8-m³ glass and stainless steel chambers with a total airflow of 2200 liters/min.
- Diet (e.g. ad libitum): NIH-07 open formula diet, Ziegler Brothers, Gardners, PA , ad libitum during times of non-exposure
- Water (e.g. ad libitum): tap water ad libitum during times of non-exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 71.5 ± 0.9°F / control chamber: 71.7 ± 0.8 °F
- Humidity (%): 55.1 ± 6.1 % / control chamber: 50.8 ± 4.8 %
- Air changes (per hr): airflow 2200 liters/min
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

not specified

Remarks on MMAD:

MMAD / GSD: no data

Details on inhalation exposure:

Pure DMA was metered from the cylinder through flow meters into the supply airstream. Test atmosphere analysis was performed at 15-min intervals by infrared spectrometry.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

analytical verification of the concentration: 175.1 ± 1.9 ppm ± SD
Pure DMA was metered from the cylinder through flow meters into the supply airstream. Test atmosphere analysis was performed at 15-min intervals by infrared spectrometry.

Duration of treatment / exposure:

2 years

Frequency of treatment:

6 h per day

Dose / conc.:

175.1 ppm (analytical)

No. of animals per sex per dose:

6 male fisher 344 rats

Control animals:

yes

Details on study design:

Groups of six male Fischer-344 rats (CDF (F- 344)/CrlBR; Charles River Breeding Laboratories, Kingston, NY) were exposed to DMA at a concentration of 175 ppm, 6 hr per day for 1,2,4, or 9 days or 2 years.

Positive control:

no

Observations and examinations performed and frequency:

no data

Sacrifice and pathology:

rats were killed by decapitation within 1 hr of the end of the exposure period.

Following the procedure described by Morgan et al. (1984), the nasal cavity was opened by rapid dissection. The dissected tissues were placed in a 145-ml stainless steel and glass observation chamber which was maintained at 36-38 °C. Humidified air was passed through the chamber at a rate of 1 liter/min during the entire observation period. The surface of the nasal mucosa was lighted with a fiber-optic light on a microscope fitted with long-working distance objectives. The mucosal surface was examined and mucus flow patterns, ciliary activity, features of nasal structure, and abnormalities of mucociliary function were recorded manually on maps of the nasal passages. Video recordings were made at 12 pre-selected locations for subsequent video analysis.
Maps of the rat nasal passages were used to record times of the various recordings as well as observations on the nature and extent of defects in mucociliary function. Ciliastasis was defined as absence of visible ciliary beat. Mucus flow rates were determined from video recordings by timing the travel of particles in the mucus epiphase over a calibrated distance on the monitor.
Following mucus flow studies, tissues were fixed in 10 % neutral buffered formalin and processed to paraffin, and 6-µm sections were cut, stained with haematoxylin and eosin, and examined by light microscopy.

Statistics:

The statistical significance of flow rate data was assessed by one-way analysis of variance. Dunnett’s multiple range test with a pooled estimate of variance was used to compare each treatment group to the appropriate control group. A nominal statistical significance level of 0.05 was selected. Because there was a difference between flow rates of acute and chronic control animals, data from treated animals were compared with the data of the respective controls. A computer software package (RS/ 1, BBN Research System, Cambridge, MA) was used for the statistical analyses.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see under other information on results

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see under other information on results

Remarks on result:

not measured/tested

Critical effects observed:

not specified

DMA-exposed animals: chronic study. Animals exposed chronically to DMA exhibited the same anatomical and functional changes described for chronic control animals. Treatment-induced destruction of nasal tissues included loss of the anterior third of the nasoturbinate and the anterodormucus flow patterns were found in association with these areas of tissue destruction, as tissue loss prevented normal flow in these areas, with mucus bypassing affected regions. Other alterations of mucus flow patterns included reversal of flow on the lateral wall in the region posterior to the dorsal margin of the maxilloturbinate, and in the affected areas the direction of ciliary beat was also modified to the direction of mucus flow. In one animal, the naso- and maxilloturbinates were fused and a thick mucus stream was seen to flow from one turbinate to the other across the point of fusion. Mucus flow rates were generally increased over those of chronic control animals. In four out of six animals in the chronic study mucociliary activity resembling that in areas lined by respiratory epithelium was observed as a posterior extension of ciliated epithelium into regions normally lined by olfactory epithelium. This was seen during the visual examination and was presumed to represent respiratory metaplasia.

Histopathology of controls. Control animals in the acute studies exhibited no abnormalities while rats in the chronic study had varying degrees of chronic inflammation in the nose. The latter lesions were confined to the areas of respiratory mucosa adjacent to the vestibule and comprised accumulations of subepithelial lymphocytes. This chronic inflammatory response was not associated with disruption of the mucociliary apparatus in affected areas.

DMA-exposed animals: chronic study. Lesions in rats exposed to DMA for 2 years were very similar to those reported previously for rats exposed for 6 months (Buckley et al.,1985). Only those changes which involve the mucociliary apparatus will be described. Again there was good correlation between the results of functional studies and histopathology. Lesions were most severe in the anterior regions of the nose and comprised focal or regional squamous metaplasia, with the normal respiratory epithelium being replaced by a stratified squamous epithelium. This response was associated with chronic active inflammation and occurred in regions with complete loss of mucociliary function. More posteriorly, the only consistent response to exposure was moderate to severe goblet cell hyperplasia, which was most prominent on the posterior lateral wall, a region which exhibited reversal of mucus flow. Respiratory metaplasia seen in four out of six animals in the mucociliary function study was characterized by posterior extension of ciliated respiratory epithelium into regions normally lined by olfactory epithelium. Histologically, these regions resembled normal respiratory epithelium except for the absence of goblet cells.

Conclusions:

The present study confirmed the previously reported respiratory metaplasia seen in the olfactory region of the nose following chronic exposure of rats to DMA (Buckley et al., 1985).

Executive summary:

The present 2 year study with dimethylamine in which male rats were dosed at 175.1 ppm has demonstrated that areas which exhibit histologic evidence of respiratory metaplasia display the ciliary activity and mucus flow characteristics associated with areas normally lined by respiratory epithelium.

It is interesting that metaplasia to a respiratory-type epithelium occurs at both extremities of the respiratory tract. However, it remains to be determined whether these responses are protective in nature or whether similar mechanisms are responsible for these changes in the nose and lung.

A number of findings were reported here for acute DMA exposures, which were not apparent in animals exposed for 6 or 12 months (Buckley et al., 1985), including focal degeneration of the squamous epithelium in the nasal vestibule and extensive vacuolation of both the respiratory and olfactory epithelia in the anterior nasal passages. Presumably, despite continued exposure, there was considerable repair in the regions lined by squamous epithelium and resolution of the vacuolation seen in the respiratory and olfactory epithelia, following chronic exposure. In the respiratory epithelial-lined regions of the anterior nasal cavity, acute epithelial degeneration led to squamous metaplasia in chronically exposed animals in both the present study and the study reported by Buckley et al. (1985) presumably as a protective or adaptive response.

It is of interest that despite severe tissue destruction in the anterior nose following a single 6-hr exposure, the nasal lesions exhibited very little evidence of progression, even after 2 years of exposure. These findings indicate a possible regional susceptibility to DMA toxicity or a degree of adaptation by the rat to continued DMA exposure.

Reference 4

Endpoint:

chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

It is considered justified to utilise information on this substance in a read across approach since, like the registered substance, it is an aliphatic amine with saturated alkyl groups. Furthermore, dimethylamine is considered to be a potential metabolite of the registered substance.

Reason / purpose for cross-reference:

read-across source

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Conclusions:

The present study confirmed the previously reported respiratory metaplasia seen in the olfactory region of the nose following chronic exposure of rats to DMA (Buckley et al., 1985).

Executive summary:

It is considered justified to utilise information on dimethylamine in a read across approach since, like the registered substance, it is an aliphatic amine with saturated alkyl groups. Furthermore, dimethylamine is considered to be a potential metabolite of the registered substance.

The present 2 year study with dimethylamine in which male rats were dosed at 175.1 ppm has demonstrated that areas which exhibit histologic evidence of respiratory metaplasia display the ciliary activity and mucus flow characteristics associated with areas normally lined by respiratory epithelium.

It is interesting that metaplasia to a respiratory-type epithelium occurs at both extremities of the respiratory tract. However, it remains to be determined whether these responses are protective in nature or whether similar mechanisms are responsible for these changes in the nose and lung.

A number of findings were reported here for acute DMA exposures, which were not apparent in animals exposed for 6 or 12 months (Buckley et al., 1985), including focal degeneration of the squamous epithelium in the nasal vestibule and extensive vacuolation of both the respiratory and olfactory epithelia in the anterior nasal passages. Presumably, despite continued exposure, there was considerable repair in the regions lined by squamous epithelium and resolution of the vacuolation seen in the respiratory and olfactory epithelia, following chronic exposure. In the respiratory epithelial-lined regions of the anterior nasal cavity, acute epithelial degeneration led to squamous metaplasia in chronically exposed animals in both the present study and the study reported by Buckley et al. (1985) presumably as a protective or adaptive response.

It is of interest that despite severe tissue destruction in the anterior nose following a single 6-hr exposure, the nasal lesions exhibited very little evidence of progression, even after 2 years of exposure. These findings indicate a possible regional susceptibility to DMA toxicity or a degree of adaptation by the rat to continued DMA exposure.

Reference 5

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

other: US EPA Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation; Human and Domestic Animals, addendum 10, Series 81, 82 and 83; EPA 540/09-91- 123

Deviations:

yes

Remarks:

Additional modifications utilised to accommodate the logistics of exposure and evaluation. Each neurotoxicity endpoint was designed to evaluate a different aspect of potential neurotoxicity (Tilson, 1987; Moser and MacPhail, 1990).

Principles of method if other than guideline:

Further references:
Moser, V.C. and MacPhail, R.C. 1990. Comparative sensitivity of neurobehavioral tests for chemical screening. Neurotoxicology 11: 285-291.
Tilson, H.A. 1987. Behavioral indices of neurotoxicity: What can be measured? Neurotoxicol. Teratol. 9: 427-443.
Tilson, H.A. 1993. Neurobehavioral methods used in neurotoxicological research. Toxicol. Lett. 68: 231-240.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Hollister/ Kingston, USA
- Age and mean weight at study initiation: Male Sprague-Dawley rats designated for schedule controlled operant behaviour (food-restricted animals) testing were received from Charles River Hollister (Hollister, California) and were 125 days of age at the initiation of exposures. As these animals were food-restricted for several weeks, they weighed 304 ± 12 g (mean ± SD). Male and female Sprague-Dawley rats designated for the other neurotoxicity endpoints (ad libitum-fed animals) were received from Charles River Kingston and were 60 days of age and weighed 266 ± 11 g (males) or 205 ± 10 g (females) at the initiation of exposures. The variability in body weight of individual animals in the selected population did not exceed 20% of the mean for each sex.
- Housing: individually in stainless-steel, wire-mesh cages
- Diet and water: ad libitum-fed animals were maintained on Certified Rodent Diet whereas food-restricted animals were fed daily amounts of 13 grams of Certified Rodent Diet. Water was available ad libitum from an automatic watering system except during operant behaviour sessions, motor activity testing, and exposures.

ENVIRONMENTAL CONDITIONS
not reported

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

not relevant

Details on inhalation exposure:

The test atmosphere was generated by metering the test substance into glass distillation columns packed with glass beads. Filtered, compressed air was passed through the glass bead-packed columns to evaporate the test substance. The distillation columns were heated to approximately 50 °C to enhance vaporization. The oxygen content of the chamber exposure atmosphere was at least 19.0 %. The total airflow was set at 12–14 air changes per h. The temperature and humidity were maintained at 20.6–24.7 °C and 36.7–68.7 %, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber vapour concentrations were determined at least once each hour with a infrared gas analyser set at a wavelength of 3.38 µm.

Duration of treatment / exposure:

6 h

Frequency of treatment:

5d/week = 65 exposure days within 13 weeks

Dose / conc.:

500 ppm

Remarks:

(ca. 2.35 mg/L analytical conc.)

Dose / conc.:

1 500 ppm

Remarks:

(ca. 7.05 mg/L analytical conc.)

Dose / conc.:

3 000 ppm

Remarks:

(ca. 14.1 mg/L analytical conc.)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The study consisted of two sets of animals, male and female ad libitum fed Sprague-Dawley (SD) rats designated for functional observational battery, motor activity, and neuropathology endpoints (FOB/MA/NP) and food-restricted male (SD) rats which were designated for schedule-controlled operant behavior (SCOB).

Animals were distributed into 4 groups of 30 animals each. Ten males and 10 females from each treatment group were designated for FOB/MA/NP testing while another 10 males from each treatment group were utilized for SCOB testing. Control (0 ppm) and high-concentration groups contained an additional 10 animals (5 males and 5 females) for possible post-exposure/recovery evaluation. Exposures were conducted 6 hr/day, 5 days/wk for 65 exposures over 13 consecutive weeks for food-restricted animals assigned to the SCOB group. Ad-libitum-fed animals assigned to the FOB/MA/NP group were exposed for 65 exposures over 14 weeks; the extra week of exposures was required because animals were not exposed on days when the FOB and MA tests were conducted.

Concentrations were selected based on the results of a two-week repeated exposure study in which animals were exposed to 0, 750, 1500, or 3000 ppm nBA. A concentration of 3000 ppm was selected as an exposure concentration that would produce overt signs of toxicity and 500 ppm was selected as an exposure concentration that was expected to have no effect. An exposure concentration of 1500 ppm was selected as the intermediate exposure concentration .

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS
Animals were observed each hour during exposure. Tapping sounds were made on the outside of the chamber to assess the animals' response to stimulation. Before and after exposure, each animal was handled and examined in detail for clinical signs of toxicity. On weekends, cage side observations were conducted.

BODY WEIGHT
For ad libitum-fed animals, body weights were measured weekly (prior to exposure) and on days the functional observational battery tests were performed. For food-restricted animals, body weights were measured daily prior to each schedule-controlled operant behaviour session.

NEUROBEHAVIOURAL EXAMINATION
Motor Activity Measurement
Motor activity for ad libitum-fed animals was measured from Tuesday through Friday of the week prior to study start (Week-1), and during Weeks 4, 8, and 13 of the study. Horizontal motor activity was measured by counting photobeam breaks at 10-minute intervals for a total of 60 minutes using an automated cage rack photobeam activity system. Following motor activity measurements, the animals were placed into their home cages for at least 30 minutes prior to conducting the functional observational battery. Ad libitum-fed animals were divided into 1 of 4 replicate groups containing 5 to 7 animals from each treatment group per sex. One replicate was tested each day at the same time of day (early morning). Animals were not exposed on the days during which motor activity was measured.

Functional Observational Battery
A functional observational battery was conducted using ad libitum-fed animals during the week prior to study start (Week -1), and during Weeks 4, 8, and 13 of the study. Animals were evaluated in random order with both observer and recorder blind to treatment. The numbers of vocalizations, urinations, and defecations during a 3-minute period of open field activity were recorded. The animals were observed, and their behaviour or condition was scored based on the system described by O'Donoghue (1996: Clinical neurologic indices of toxicity in animals. Environ Health Perspect 1996; 104 (Suppl 2): 323-33 0). A quantitative assessment of forelimb and hindlimb strength grip strength was performed with an apparatus equipped with a digital push-pull gauge. The rat was placed on a rectangular screen and then lifted upwardly until it released its grip. The grip strength test was repeated for a total of three readings. The mean of the three readings was reported as the grip strength measurement. A quantitative assessment of foot splay was performed by releasing a rat 32 cm above a bench in a horizontal position and allowing it to drop. Foot splay was determined by measuring the distance between marks caused by ink placed on the outside digit of each hindfoot with a water-soluble marker. Animals were divided into the same replicates used for motor activity measurements, but observed at least 30 minutes after returning to their home cage. Animals were not exposed on the days during which an FOB was conducted.

Schedule-Controlled Operant Behaviour
The equipment consisted of operant chambers (Model E10-09) in isolation cubicles with a house light, three cue lights (over the lever), one lever (on the right side), a 2.5 kHz tone, and a food pellet dispenser. Test sessions were conducted in a room separate from the housing room. Bedding material was placed in trays under the operant chambers and was changed daily. Food pellet dispensers were cleaned weekly. Operant chambers were cleaned weekly with an Alconox solution after which the chambers were wiped down with 70 % isopropyl alcohol. The chambers were vacant and open for at least 24 hours after cleaning. Animals were motivated to press a lever by restricting their food to 13 grams/day and reinforcing lever presses with 45-mg food pellets. A multiple schedule of four fixed-ratio components followed by two fixed-interval components was used. The lights above the lever served as the cue for the FR component, and a 2.5 kHz tone at 70 ± 2 dB served as the cue for the FI component. The sessions were 47 minutes long and were conducted once daily, 5 days per week at the same time each day 1-2 h prior to exposure. The animals acquired the behaviour over the course of several weeks prior to the start of the study using a weekly progression from continuous FR to FR5 (fixed ratio 5 lever presses) followed by FR10 (fixed ratio 10 lever presses) schedules, then adding a F160 (fixed interval 60 seconds) component. The FR and FI components were then increased to the final multiple FR:FI schedule. Stable behaviour (coefficients of variation for Fl index of curvature and Fl response rate of <20 %) were demonstrated prior to the initiation of exposures.
Operant behaviour was measured for four consecutive days (Tuesday-Friday) prior to the first exposure to establish baseline response rates. Animals were divided into two replicates (20 per replicate) evenly distributed across exposure groups and equipment. Parameters calculated were FR run rate, post-reinforcement pause duration, F1 response rate, and index of curvature. The FR run rate is defined as the number of lever presses per minute during the time interval from the first lever press to the 20th lever press. Lever presses that occurred during the first 250 msec were not counted (overflow responses). The post-reinforcement pause duration is defined as the time interval from the reinforcement of the last FR run to the next lever press. If overflow responses occurred during this time, the pause duration was the interval from the last overflow response to the first lever press of the next FR run. The FI response rate is defined as the number of lever presses per minute during the time interval from the start of the F1 run until the reinforcement. The index of curvature is the ratio of the area under the response-time curve within each of the 5 bins to the area under the hypothetical straight line of constant-rate responses. Weekly means of FR run rates, pause duration, FI response rates, and FI index of curvature values were compared with the baseline values, and the percent of baseline was calculated for each animal and group. The data from Weeks 4, 8, and 13 were evaluated for evidence of neurotoxicity. Data were also collected for two weeks following the cessation of exposure.

Sacrifice and pathology:

At the end of the exposure period, five ad libitum fed animals per sex were randomly selected from each group for perfusion and neurohistopathology evaluation. The remaining ad libitum-fed animals and all the food restricted animals were humanely killed, and the carcasses were discarded. Animals designated for neuropathologic examination were anesthetized with sodium pentobarbital containing heparin (10 % by volume) and perfused through the ascending aorta. The perfusates were 4 % paraformaldehyde followed by 5 % glutaraldehyde, both in 0.1 M phosphate buffer, pH 7.4, at VC. The following tissues were collected from all perfused animals: brain (including the forebrain, cerebrum, midbrain, cerebellum, pons, medulla oblongata); spinal cord swellings with dorsal and ventral roots (cervical and lumbar); dorsal root ganglia (cervical and lumbar); sciatic nerve (both hindlimbs at mid-thigh and sciatic notch); and tibial nerve (both hindlimbs including branches to the calf musculature).
The peripheral nerves from the left leg were fixed for an additional period of at least 2 hours in 5 % glutaraldehyde and stored in sodium phosphate buffer until processed. After processing was completed, the remaining peripheral nerve tissue from the left leg was stored in neutral lbuffered 10 % formalin.
Brain and spinal cord sections from the high concentration and control groups were processed by routine neuropathological techniques for paraffin embedment, haematoxylin-eosin staining, and light microscopic examination. The left sciatic and tibial nerves, dorsal and ventral roots, and the dorsal root ganglia from the high concentration and control groups were processed and embedded in glycol methacrylate, sectioned at 2 µm, and stained with 1 % toluidine blue.

Statistics:

Body weight data were evaluated using the following statistical tests: Bartlett’s test (p ≤ 0.01) to test for equality of variances, one-way analysis of variance (ANOVA, p ≤ 0.05), and Duncan’s multiple range test (p ≤ 0.05) for statistical significance. Continuous data from FOB and MA testing were analysed by repeated measures of analysis of variance (SAS Institute, Cary, NC, USA). Baseline (pre-exposure) values were subtracted from test-day (weeks 4, 8, 13) values to normalise the animal to animal variance in absolute scores. Time points indicating significant changes were further analysed by a Fisher’s Exact Test. A probability of p < 0.05 (two tailed) was used to determine significance which was not corrected for the number of comparisons conducted. If the Bartlett’s test demonstrated unequal variances, then the data were evaluated with a Kruskal-Wallis H-test and Mann-Whitney U-test.
SCOB data were analysed as mean weekly percent of baseline values using ANOVA (p < 0.05) and Dunnett’s t-test (p < 0.05) to indicate statistical significance, and the absolute values were analysed for trends using a multivariate, repeated-measures analysis of variance. SCOB data for weeks 13 and 15 were compared using a paired Student’s t-test and a probability of p < 0.05 (two-tailed) was used to indicate significance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals exposed to 3000 ppm (ca. 14.1 mg/L) exhibited less movement, decreased alertness, and slower response to tapping on the exposure chamber wall (reduced reactivity) during exposure compared with activity levels exhibited by control animals. Animals exposed to 1500 ppm ( ca. 7.05 mg/L) appeared normal for the first 5 hours of Day 0 (the first day of exposure) and the first hour or two of Days 1 and 2, and thereafter exhibited reduced reactivity of generally minimal severity for the remainder of the daily exposure periods. Control and 500 ppm animals appeared normal during exposure. There was no apparent difference in the clinical conditions of ad libitum-fed and food-restricted animals. There were no signs of neurobehavioural effects or systemic toxicity immediately after exposure (30-60 minutes following the cessation of exposure).

Mortality:

no mortality observed

Description (incidence):

No spontaneous mortality occurred during the study, but one ad libitum-fed control male was euthanised in a moribund condition due to poor physical condition and excessive weight loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights for the 3000 ppm groups were generally 15-19 % lower than the mean body weight of the control group and were significantly lower than the control group beginning on Day 7 for male rats and Day 14 for female rats. Overall weight gains for the 3000 ppm group were 64 and 59 % of weight gains for the control group (males and females, respectively). Overall, mean body weights for the 1500 ppm group were 9 % lower than the mean body weights of the control group. However, mean body weights for the female 1500 ppm group were significantly lower than the control group on Days 42, 49, 70, 77, 84, 91, and 98. Mean body weights for the 500 ppm group were comparable to the control group throughout the study, and no statistically significant differences were noted.
No differences in mean body weight of food restricted animals were noted among groups.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

FUNCTIONAL OBSERVATIONAL BATTERY
In male rats, significant time-treatment interactions were found in the severity scores of nasal discharge (a possible autonomic parameter). Pre-exposure baseline severity values of nasal discharge were substantially higher for the control group than for the 500 ppm group. As a result, mean severity scores of nasal discharge during Weeks 4 and 8, which were corrected for baseline, were significantly higher in the 500 ppm group than in the control group. This finding was not considered to be toxicologically or neurobehaviorally significant or related to exposure to butyl acetate. No other time-treatment interactions were observed. In female rats, no time-treatment interactions were observed.

MOTOR ACTIVITY
In general, the mean total motor activity counts and total ambulations for all male groups were higher during Week 4 than at other times although only slightly higher (11 % for motor activity counts). Repeated-measures analysis indicated a time-treatment interaction in total motor activity counts with the baseline-corrected mean total motor activity for the 3000 ppm male group significantly higher than for the control group at Week 4. Mean total motor activity counts for all male groups were closer to baseline values during Weeks 8 and 13 and no significant differences were observed among groups. Given the slight difference between the counts for the 3000 ppm and control groups, and the lack of a difference at Weeks 8 and 13, the significance of the finding at Week 4 is questionable. In addition, graphic representation of motor activity during the 60 minute measurement period indicates that the activity was similar for all groups during Weeks 4 and 13. In contrast to total motor activity counts, no time-treatment interactions were observed in total ambulations for male groups. For female groups, the mean total motor activity counts and total ambulations were also higher during Week 4 than at other times, but there were no statistically significant differences. Mean total motor activity counts for female rats were also closer to baseline values during Weeks 8 and 13.

SCHEDULE-CONTROLLED OPERANT BEHAVIOUR
Baseline schedule-controlled operant behaviour values were comparable among groups prior to the initiation of exposure, and no statistically significant difference in any schedule-controlled operant behaviour parameter was noted among groups prior to exposure or at any of the four specified time periods. In general, FR run rates increased 20-37 % over the course of the study and appeared to be unaffected by exposure to the test substance. Run rates increased in each group approximately the same amount relative to baseline. Changes in pause duration were variable over the course of the study, with the control and 3000 ppm groups having longer durations relative to baseline, and the 500 and 1500 ppm groups having shorter durations relative to baseline. These differences in pause duration among groups appear to reflect the number of animals in each group with pause durations of less than or greater than 6.0 seconds. In general, pause times of less than 6 .0 seconds at baseline became longer over the course of the study, whereas pause times of greater than 6.0 seconds became shorter over the course of the study. These trends appeared to be unaffected by exposure to the test substance. Under the FI schedule, the activity of the control group remained relatively constant throughout the study. Mean overall response rates for the control group were within 10 % of baseline during Weeks 4, 8, and 13; by contrast, Fl response rates for the treated groups decreased over the course of the study to 70 % of baseline. Decreases in mean response rates were first observed in the 3000 ppm group at Week 4, but by Week 13, all treated groups had lower mean response rates relative to baseline. Index of curvature values reflect the FI response rates in that index of curvature values for the control group remained near baseline throughout the study, and index of curvature for the treated groups increased. There were no apparent concentration-related differences among groups. There were no significant concentration-time interactions in any SCOB parameters, and there were no significant changes in SCOB parameters following cessation of exposure relative to the last week of exposure.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic evaluations of sections from the brain, spinal cord (cervical and lumbar regions), dorsal and ventral spinal roots, dorsal root ganglia, sciatic nerve, and tibial nerve of animals in the control and 3000 ppm groups did not indicate any treatment-related effects.

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

ca. 2.35 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

other: corresponding to 500 ppm

Critical effects observed:

not specified

Conclusions:

Under the conditions of the study the No Observed Adverse Effect Concentration was determined to be 500 ppm (ca. 2.35 mg/L).

Executive summary:

Neurotoxicity investigations were undertaken following repeated dose exposure of the test material to groups of Sprague-Dawley rats. The rats were exposed (whole body) to test material (vapour exposure) at 500, 1500, 3000 ppm for 6 hours a day, 5 days per week, for 13 weeks.

Under the conditions of the study there was no evidence of cumulative neurotoxicity based on a functional observational battery, motor activity measurements, neurohistopathology, and schedule-controlled operant behaviour endpoints. The No Observed Adverse Effect Concentration was determined to be 500 ppm (ca. 2.35 mg/L).

Reference 6

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

It is considered justified to utilise information on this substance in a read across approach since its metabolite (n-butanol) is also a potential metabolite of the registered substance.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

ca. 2.35 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

other: corresponding to 500 ppm

Critical effects observed:

not specified

Conclusions:

Under the conditions of the study the No Observed Adverse Effect Concentration was determined to be 500 ppm (ca. 2.35 mg/L).

Executive summary:

It is considered justified to utilise information on n-butylacetate in a read across approach since its metabolite (n-butanol) is also a potential metabolite of the registered substance.

Neurotoxicity investigations were undertaken following repeated dose exposure of the test material to groups of Sprague-Dawley rats. The rats were exposed (whole body) to test material (vapour exposure) at 500, 1500, 3000 ppm for 6 hours a day, 5 days per week, for 13 weeks.

Under the conditions of the study there was no evidence of cumulative neurotoxicity based on a functional observational battery, motor activity measurements, neurohistopathology, and schedule-controlled operant behaviour endpoints. The No Observed Adverse Effect Concentration was determined to be 500 ppm (ca. 2.35 mg/L).

Reference 7

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

EPA OTS 798.2450 (90-Day Inhalation Toxicity)

Deviations:

not specified

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Kingston, USA
- Age at study initiation: ca. 60 d
- Mean weight at study initiation: 271± 7 g (males); 215 ± 8 g (females); the variability in body weight of individual animals in the selected population did not exceed 20 % of the mean for each sex
- Housing: individually in stainless-steel, wire-mesh cages
- Diet: Certified Rodent Diet ad libitum
- Water: filtered municipal tap water

ENVIRONMENTAL CONDITIONS
not reported

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

The test atmosphere was generated by metering the test substance into glass distillation columns packed with glass beads. Filtered, compressed air was passed through the glass bead-packed columns to evaporate the test substance. The distillation columns were heated to approximately 50 °C to enhance vaporization. The oxygen content of the chamber exposure atmosphere was at least 19.0 %. The total airflow was set at 12–14 air changes per h. The temperature and humidity were maintained at 20.6–24.7 °C and 36.7–68.7 %, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber vapour concentrations were determined at least once each hour with an infrared gas analyser set at a wavelength of 3.38 µm.

Duration of treatment / exposure:

6 h

Frequency of treatment:

5d/week = 65 exposure days within 13 weeks

Dose / conc.:

500 ppm

Remarks:

(ca. 2.35 mg/L analytical conc.)

Dose / conc.:

1 500 ppm

Remarks:

(ca. 7.05 mg/L analytical conc.)

Dose / conc.:

3 000 ppm

Remarks:

(ca. 14.1 mg/L analytical conc.)

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A concentration of 3000 ppm was selected as an exposure concentration that would produce overt signs of toxicity, and 500 ppm was selected as an exposure concentration that was expected to have no effect. An exposure concentration of 1500 ppm was selected as the intermediate exposure concentration (based on the results of a 2-week repeated exposure study in which animals were exposed to 0, 750, 1500 or 3000 ppm nBA).

Observations and examinations performed and frequency:

OBSERVATIONS
Body weights and feed consumption were measured weekly prior to exposure. Animals were fasted the day prior to necropsy. Fasted body weights were measured after exsanguination, but prior to necropsy. This procedure is standard practice in the laboratory because it allows for blood collection from the posterior vena cava while the animal is under anaesthesia, and reduces the variability in the ratio of body and organ weight due to differences in body fluids. Before and after exposure, each rat was removed from its cage and examined. Cage-side observations were conducted once a day on weekends. Observations included, but were not limited to, examination of the hair, skin, eyes and mucous membranes, motor activity, faeces, urine, respiratory system, circulatory system, autonomic nervous system, central nervous system, and behaviour patterns.

HAEMATOLOGY and CLINICAL CHEMISTRY
Animals were fasted beginning after their last exposure. The following day, animals were anesthetized with Metofane, and blood was collected from the posterior vena cava. The blood was placed into vacutainer tubes and allowed to clot for analyses of serum. Other tubes containing an anticoagulant were used for analyses of whole blood samples. Blood smears were also prepared for blood cell counts. Following blood collection, the animals were killed humanely by exsanguination under anaesthesia. Animals were bled and euthanatized in random order based on a computer-generated list.
Whole blood samples were analyzed for red blood cell count, total white blood cell counts, haemoglobin, haematocrit and red blood cell indices using an haematology analyzer. Prothrombin time was measured using a Fibrosystems analyzer. Slides with blood smears were stained and examined for cellular morphology, differential white blood cell count and platelet count. Slides with blood smears were stained and examined for cellular morphology, differential white blood cell count and platelet count.
Serum samples were analyzed for total protein, total bilirubin, calcium, phosphorous, urea nitrogen, creatinine, glucose, gamma-glutamyl transferase, aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase and alkaline phosphatase using a serum analyzer. Albumin concentration and isozyme profile were determined using a Gel Electrophoresis System. The albumin/globulin ratio was calculated from total protein and albumin concentrations. Serum sodium and potassium concentrations were determined using a Photometer and serum chloride concentration was measured using a chloride Analyzer.

OPHTHALMOSCOPY
All rats were examined by a veterinarian for retinal and corneal lesions prior to the start of the study using a direct ophthalmoscope. During the last week of exposure, animals from the control and high-concentration groups were re-examined. Because no changes were detected in the eyes of the high-concentration animals, the animals from the low- and mid-concentration groups were not re-examined.

Sacrifice and pathology:

On day 30, five male and five female rats from each group were anaesthetized with Metofane and blood was collected for clinical pathology; these animals were then euthanatized and the carcasses were discarded.

After 13 weeks of exposure, animals were fasted overnight. The following day, animals were anaesthetized with Metofane, and blood was collected from the posterior vena cava. The animals were then exsanguinated and weighed. Wet weights of the liver, kidneys, testes or ovaries, spleen, adrenal glands, lungs and brain were recorded for all animals at necropsy. Paired organs were weighed together, except for the testes, which were weighed individually. All tissues listed in the US EPA Health Effects Testing Guideline for Inhalation Toxicity (40 CFR 798.2450) were collected and preserved in 10 % buffered formalin (pH 7.4). The capsule of the right testis was pricked with a 22-gauge needle and preserved in Millonig’s fixative (10 % neutral buffered formalin with phosphate buffered saline, pH 7.4).
All tissues listed below were embedded in paraffin, sectioned at 5 µm, and stained with haematoxylin and eosin (H&E). The nasal passages were decalcified prior to being embedded and sectioned. The lungs were sectioned along a plane allowing visual examination of the major bronchi and bronchioles. The right testis was embedded in glycol methacrylate, sectioned, and stained with H&E and periodic Schiff’s reagent. All tissues except for the brain, spinal cord and peripheral nerve were examined microscopically from the control and high-concentration groups. Nervous tissue from animals exposed simultaneously was evaluated in the neurotoxicity study (CMA 1996b). The lungs, nasal passages, thymus (males only), stomach (females only) and gross lesions were examined from the mid- and low concentration groups.

Other examinations:

The left testis and left epididymis of each male rat were placed into individual bags and frozen at -25 °C for sperm counts. Samples were shipped to Research Triangle Institute (Research Triangle Park, NC, USA) for analysis. The frozen tissues were weighed to assess the effect of freezing, and homogenized according to the procedure described in Fail et al. (1991). The number of elongated spermatids (testes) or spermatozoa (epididymis) were counted. The results are expressed per gram tissue weight.

Statistics:

Body weight, feed consumption, serum chemistry, haematology, organ weight and sperm count data were evaluated using the following statistical tests: Bartlett’s test (P <= 0.01), one-way analysis of variance (ANOVA) (P <= 0.05), and Duncan’s multiple range test (P <= 0.05) or Dunnett’s test to indicate statistical significance. If the Bartlett’s test indicated unequal variances, the data were evaluated using the Kruskal–Wallis H-test and the Mann–Whitney U-test.
A probability of P <= 0.05 (two-tailed) was used to determine significance. If the Bartlett’s test indicated unequal variances, the data were evaluated using the Kruskal–Wallis H-test and the Mann–Whitney U-test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals exposed to 3000 ppm (ca. 14.1 mg/L) had reduced activity levels during exposure that were of generally minor severity. Reduced activity was defined as less movement, decreased alertness, and slower response to tapping on the chamber wall compared with activity levels exhibited by control animals. Signs of diarrhoea and red discoloration on the chin hair were also observed. Animals exposed to 1500 ppm (ca. 7.05 mg/L) appeared normal for the first 5 h of day 0 and the first hour or two of days 1 and 2, then exhibited reduced activity of generally minimal severity for the remainder of the daily exposure periods. Reduced activity of minimal severity was generally seen throughout daily exposures thereafter. Control and 500 ppm (2.350 mg/L) animals appeared normal during exposure.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in any of the treated groups during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights for the 1500 and 3000 ppm groups (ca. 7.05 and 14.1 mg/L) were significantly lower than the control group for most of the study. Overall weight gains for the 3000 ppm group were 62 and 78 % of weight gains for the control group (males and females, respectively), while overall weight gains for the 1500 ppm groups were 77 and 70 % of the control group (males and females, respectively).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption for the 3000 ppm groups were significantly lower than for the control group throughout the study for male rats and at all intervals except days 84 and 91 for female rats. Mean feed consumption values for the 1500 ppm groups were significantly lower than the control group for the majority of the study, and sporadically lower for the 500 ppm groups compared with the control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related ophthalmologic changes were observed.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No effects considered as biologically relevant were observed.
No significant differences in haematologic parameters were seen after 30 days on test. Significantly higher mean erythrocyte counts, haemoglobin concentration and haematocrit values were observed for the 3000 ppm male and female rats after 90 days on test compared with the control groups . The mean eosinophil percentage for male 3000 ppm rats was also significantly higher than for the control group. All the values were within normal limits for rats of this age and for the age and strain of the animals used in this laboratory, and none of the differences were considered biologically significant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

After 30 days on test, mean sodium concentrations for the male and female 3000 ppm groups were significantly lower than for the control group. The differences were slight (ca. 1 Meq/L), however. The mean chloride concentration for the 1500 ppm male group was significantly lower compared with the control group. However, this difference was also small (< 4 Meq/L). No other differences in serum chemistries were seen among groups. After 90 days on test, mean albumin and total protein concentrations for the 3000 ppm female group were significantly lower than for the control group. Mean sorbitol dehydrogenase activity for the 1500 ppm male group was significantly higher than for the control group. These changes were not considered to be toxicologically meaningful, and no other differences in serum chemistry were observed among groups.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Terminal body weights measured after exsanguination were significantly lower for the 1500 and 3000 ppm male and female groups compared with the control group. Absolute weights of the liver, kidneys and spleen reflect this reduced body weight. Liver and spleen weights for the 1500 and 3000 ppm male and female groups were significantly lower than for the control groups. Absolute kidney weights for the 1500 ppm female and 3000 ppm male and female groups were also significantly lower than for the control groups. However, relative organ weights (to body weight) for these organs were not significantly different with the exception of the spleen-to-body weight ratio for the 3000 ppm male group, which was significantly lower than for the control group.
Reduced body weight was also reflected in the significantly lower absolute brain weight for the 3000 ppm male group and significantly higher brain-to-body weight for the 1500 ppm female and 3000 ppm male groups compared with their respective control groups. In addition, testes-to-body weights for the 1500 and 3000 ppm male groups and the relative lung (to body weight) weight for the 3000 ppm male group were significantly higher than for the control group. Adrenal gland-to-body weight ratios for the 1500 ppm female and 3000 ppm male and female groups were significantly higher than for the respective control groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Exposure-related changes were observed in the nasal passages and stomachs of 1500 and 3000 ppm rats. All male and female 3000 ppm rats and 4/10 male and 6/10 female 1500 ppm rats had necrosis of the olfactory epithelium. Degeneration of the olfactory epithelium was observed along the dorsal medial meatus. Degeneration and regeneration of the epithelium was seen along the third ethmoturbinate and several others. The lesion was characterised by karyorrhexis, pyknosis and depletion of olfactory epithelium cells. The severity of the olfactory lesion was mild to moderate for the 3000 ppm group and minimal to mild for the 1500 ppm group. Olfactory epithelium was replaced in some areas by transitional or respiratory epithelium. A few (3/10) 3000 ppm female rats had acute inflammation and degenerative lesions (erosion) of the stomach mucosa (glandular vs forestomach). The severity was minimal to mild.
Lesions of this type in the stomach may be associated with swallowing of mucous containing the test substance, but due to the location of the lesion are more likely caused by stress (Glavin et al., 1991: The neurobiology of stress ulcers. Brain Research Reviews 16, 301–343). An occasional 3000 ppm male rat had atrophy of the thymus, but this was not considered to be a direct compound related effect. Instead, this lesion was attributed to stress (Greaves and Faccini, 1992: Rat Histopathology, A Glossary for Use in Toxicity and Carcinogenicity Studies. p. 51. Elsevier Science, Amsterdam).

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

SPERM COUNTS
No dose-related or statistically significant effect on epidydimidal or testicular sperm count was observed compared with controls, although the epididymidal sperm counts for all treated groups were lower than controls.

Dose descriptor:

NOAEC

Remarks:

local & systemic

Effect level:

ca. 2.35 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 500 ppm; based on reduced body weight, food consumption and transient CNS effects; signs at necropsy of the olfactory epithelium

Critical effects observed:

no

Decreased body weight and feed consumption were noted for the 1500 and 3000 ppm groups, but there was no systemic or organ-specific toxicity. Degeneration of the olfactory epithelium at concentrations of 1500 and 3000 ppm was observed in areas of the nasal cavity that have demonstrated carboxylesterase activity (Bogdanffy, 1990: Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach. Environmental Health Perspectives 85, 177–186), but there was no evidence of pulmonary toxicity.

Conclusions:

The No Observed Adverse Effect Concentration was determined to be 500 ppm (ca. 2.35 mg/L) based on reduced body weight and feed consumption noted for the 1500 and 3000 ppm groups. However, there was no systemic or organ-specific toxicity. Degeneration of the olfactory epithelium at concentrations of 1500 and 3000 ppm was observed in areas of the nasal cavity that have demonstrated carboxylesterase activity (Bogdanffy, 1990: Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach. Environmental Health Perspectives 85, 177–186), but there was no evidence of pulmonary toxicity.

Executive summary:

The repeated dose toxicity of the test material was investigated in a study which was conducted in accordance with the standardised guideline EPA OTS 798.2450, and under GLP conditions.

During the study, groups of Sprague-Dawley rats were exposed (whole body) to test material (vapour exposure) at 500, 1500, 3000 ppm for 6 hours a day, 5 days per week, for 13 weeks.

Under the conditions of the study, the No Observed Adverse Effect Concentration was determined to be 500 ppm (ca. 2.35 mg/L) based on reduced body weight and feed consumption noted for the 1500 and 3000 ppm groups. However, there was no systemic or organ-specific toxicity. Degeneration of the olfactory epithelium at concentrations of 1500 and 3000 ppm was observed in areas of the nasal cavity that have demonstrated carboxylesterase activity (Bogdanffy, 1990: Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach. Environmental Health Perspectives 85, 177–186), but there was no evidence of pulmonary toxicity.

Reference 8

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

It is considered justified to utilise information on this substance in a read across approach since its metabolite (n-butanol) is also a potential metabolite of the registered substance.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEC

Remarks:

local & systemic

Effect level:

ca. 2.35 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 500 ppm; based on reduced body weight, food consumption and transient CNS effects; signs at necropsy of the olfactory epithelium

Critical effects observed:

no

Conclusions:

The No Observed Adverse Effect Concentration was determined to be 500 ppm (ca. 2.35 mg/L) based on reduced body weight and feed consumption noted for the 1500 and 3000 ppm groups. However, there was no systemic or organ-specific toxicity. Degeneration of the olfactory epithelium at concentrations of 1500 and 3000 ppm was observed in areas of the nasal cavity that have demonstrated carboxylesterase activity (Bogdanffy, 1990: Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach. Environmental Health Perspectives 85, 177–186), but there was no evidence of pulmonary toxicity.

Executive summary:

It is considered justified to utilise information on n-butylacetate in a read across approach since its metabolite (n-butanol) is also a potential metabolite of the registered substance.

The repeated dose toxicity of the test material was investigated in a study which was conducted in accordance with the standardised guideline EPA OTS 798.2450, and under GLP conditions.

During the study, groups of Sprague-Dawley rats were exposed (whole body) to test material (vapour exposure) at 500, 1500, 3000 ppm for 6 hours a day, 5 days per week, for 13 weeks.

Under the conditions of the study, the No Observed Adverse Effect Concentration was determined to be 500 ppm (ca. 2.35 mg/L) based on reduced body weight and feed consumption noted for the 1500 and 3000 ppm groups. However, there was no systemic or organ-specific toxicity. Degeneration of the olfactory epithelium at concentrations of 1500 and 3000 ppm was observed in areas of the nasal cavity that have demonstrated carboxylesterase activity (Bogdanffy, 1990: Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach. Environmental Health Perspectives 85, 177–186), but there was no evidence of pulmonary toxicity.

Reference 9

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

It is considered justified to utilise information on this substance in a read across approach since it is a potential metabolite of the registered substance.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

LOEC

Effect level:

0.15 mg/L air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Clinical chemistry, hematology

Critical effects observed:

not specified

Conclusions:

Under the conditions of this study the LOEC was 50 ppm, 0.15 mg/L air (analytical).

Executive summary:

It is considered justified to utilise information on this substance in a read across approach since it is a potential metabolite of the registered substance.

Sub chronic effects of butan-1-ol were studied in male rats exposed (6h/d; 5d/wk) to 0.15 or 0.31 mg/L (50 or 100 ppm) for 90 days (12 animals per dose).

During the study, there was no mortality and no evidence of clinical signs of toxicity at either exposure concentration. There were no effects on body weight or on absolute or relative (to body weight) organ weights of 7 organs examined. There was a slight but statistically significant decrease in haemoglobin concentration at both exposure concentrations (this did not appear to be dose-related) and a decrease in red blood cell count in the 100-ppm group. There were no statistically significant changes in serum enzymes or in mixed function oxidase activity, however, lipid peroxidation activity was increased at both exposure concentrations (this did not appear to be treatment-related). The authors reported a decreased performance on the rotarod used to assess neurological effects at both concentrations, which increased as the study progressed. The decrease appeared to be dose-related. There were no apparent changes in sensitivity of thermal response at either concentration.

Under the conditions of this study the LOEC was 50 ppm, 0.15 mg/L air (analytical).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

93.5 mg/m³

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

other: read-across target

Principles of method if other than guideline:

Male and female F-344 rats and B6C3F1 mice were exposed by inhalation to 0, 10, 50, or 175 ppm dimethylamine (DMA) for 6 hr/day, 5 days/week for 12 months. Groups of 9-10 male and female rats and mice were necropsied after 6 and 12 months of exposure.
The purpose of this study was to investigate the toxicity associated with chronic inhalation exposure of F-344 rats and B6C3F1 mice to DMA for 2 years. This report summarises the clinical and pathologic data found for the first 12-month period.

GLP compliance:

not specified

Limit test:

no

Species:

other: rat and mouse

Strain:

other: F-344 rats and B6C3F1 mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River breeding laboratories, Kingston, New York, USA
- Age at study initiation: 4-8 weeks
- Housing: individually in hanging stainless steel wire mesh cages in the exposure chambers
- Diet: NIH-07 open formula diet, Ziegler Brothers, Gardners, Pa.; analyzed for contaminants by Lancaster Labs, Lancaster, Pa., ad libitum during periods of non-exposure
- Water: tap water via an automatic watering system, ad libitum during periods of non-exposure
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 - 76 °F
- Humidity (%): 45 - 65 % ( real: 35 - 74 %)
- Air: airflow of 2200 liters/min
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: n.a.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: test atmospheres were generated by metering pure DMA directly from the cylinder via flowmeters (Fischer-Porter, Warminster, Pa., or Calibrating and Measuring Equipment, Inc., Manassas, Va.) into the supply air stream.
- Method of holding animals in test chamber: rats and mice were exposed and housed in 8-m3 stainless steel and glass whole body chambers operated with a dynamic airflow of approximately 2200 liters/min (HEPA-filtered room air) and at slightly subatmospheric pressure (0.2-0.3 in. of water). The cages within a rack were rotated once per week using a computer-generated randomisation procedure designed to ensure that each animal spent an equal amount of time in all areas of the chamber.

- Method of conditioning air: HEPA filter
- System of generating particulates/aerosols: DMA directly entered the air stream
- Temperature, humidity, pressure in air chamber: slightly subatmospheric pressure (0.2-0.3 in. of water).
- Air flow rate: 2200 liters/min
- Air change rate: approx. 15
- Method of particle size determination: n.a.

TEST ATMOSPHERE
- Brief description of analytical method used: IR spectrophotometer
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the test atmospheres was performed four times per hour by infrared spectrometry at a wavelength of 3.5 µm and a path length of 20.25 m (MIRAN 801, Foxboro-W&s, Norwalk, Conn.). The spectrophotometer was set to zero optical density with ultra zero air containing approximately 350 ppm Co, (Ma&son Gas, Morrow, Ga.) and adjusted to approximately 50 % relative humidity. Temperature and relative humidity were recorded hourly.

The mean time-weighted average (TWA) analytical chamber concentrations for the 12 month period, derived from daily TWA concentrations, with standard deviations and ranges were
175 ppm +/- 2.0 (167-188),
50.0 ppm +/- 1.0 (45.8-54.0), and
10.0 ppm +/- 0.3 (9.0-10.8).

The average percentage ratios of analytical/nominal concentrations (+/- standard deviations) were 81 +/- 8, 87 +/- 7, and 76 +/- 15 % for the 175-, 50-, and 10-ppm chambers, respectively, thus indicating a fairly constant loss of DMA on the chamber surfaces and animals for each chamber. The distribution of the analytical concentration of DMA in the chambers was determined to be uniform within +6 % of target concentrations.

Duration of treatment / exposure:

12 month

Frequency of treatment:

5 days/week, 6 hours/day

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

10 ppm (nominal)

Dose / conc.:

50 ppm (nominal)

Dose / conc.:

175 ppm (nominal)

No. of animals per sex per dose:

95 animals

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: dose levels were selected on the basis of preceding studies. F-344 rats exposed to 175 or 250 ppm DMA (6 hr/day, 5 days) or 500 ppm DMA (6 hr/day, 3 days) had ulcerative rhinitis, severe congestion, and squamous metaplasia in the respiratory tract.These lesions were most severe in the anterior sections of the nasal passages.

Observations and examinations performed and frequency:

The animals were observed for clinical abnormalities twice daily and were weighed once per week for the first 13 weeks, and biweekly thereafter.

Sacrifice and pathology:

After 6 and 12 months, 9-10 animals of each species, sex, and treatment group were fasted overnight and weighed just prior to necropsy. Male mice were not included in the 12-month sacrifice because of the high rate of unscheduled mortality in this group.

Animals were anesthetised with pentobarbital by i.p. injection, and blood was drawn from the heart for haematology and serum chemistry. Each animal was examined for gross abnormalities, and 45 tissues and any gross lesions were collected and placed in 10 % buffered formalin. The nasal passages were flushed, the lungs inflated, and the lumen of the gastrointestinal tract infused with formalin. The liver, kidneys, and brain were weighed. All tissues from the control and 175 ppm exposed animals and target tissues (nasal turbinates) from the 10- and 50- ppm exposed animals were processed for light microscopic examination. Tissues containing bone were placed in 10 % buffered formalin for 48 hr, desiccated, then rinsed with tap water for at least 4 hr, and replaced in formalin. Tissues were then dehydrated in graded alcohols, cleared with xylene, and embedded in paraffin wax. Transverse blocks of the nose were cut and prepared to yield histological sections at the following levels: (1) just anterior to the incisor teeth, (2) approximately one third of the distance from the posterior aspect of the incisor teeth to the incisive papilla, (3) at the incisive papilla, (4) at the crest of the second palatial ridge, and (5) at the centre of the second molar tooth. Embedded tissues were sectioned at 5 pm and stained with haematoxylin and eosin for light microscopic examination. For photography, selected tissues were removed from paraffin re-embedded in glycol methacrylate (GMA), and 2- to 3- am-thick sections were cut and stained with Lee’s methylene blue. Selected nasal sections were stained with Alcian blue at pH 2.5 for acidic mucous glycoproteins.

Other examinations:

Hematological data were collected using a Coulter S Plus II counter. Sodium and potassium determinations were performed with a flame photometer. Chloride analyses were performed with a Coming Chloride 920 M meter (Coming Scientific Inst., Mechield, Mass.). An Abbott VP clinical analyser was used for determination of serum chemistries.

Statistics:

Data for body weights, serum chemistry, haematology, and organ weights were analyzed using an analysis of variance. Dunnett’s test was employed to detect differences between control and treatment groups (Steel and Torrie, 1980). Red blood cell morphology and histopathological findings were analyzed using the Kolmorgomov-Smimov test (Daniel, 1978). In all cases, the preselected significance level was p ≤ 0.05.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

significant loss at 175 ppm ( 90 % of control)

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

decreased platelet counts (175 ppm), increased counts of atypical lymphocytes in females (175 ppm), decreased mean red blood cell volume (females, 175 ppm)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

decreased protein (175 ppm), increased alkaline phosphatase (females, 175 ppm)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

(1) LESIONS IN RESPIRATORY EPITHELIUM
The lesions in the respiratory area were most severe on the anterior septum, just posterior to the vestibule, and on the free margins of the naso- and maxilloturbinates, with lesser involvement of the lateral wall. In the 175 ppm exposure group, there was variable destruction of the anterior portions of the naso- and maxilloturbinates, and fenestration of the nasal septum. In areas of destruction of the turbinates and septum, the surface was covered by nonkeratinizing squamous epithelium. Evidence of both acute and chronic inflammatory response included focal to diffuse mucosal and submucosal infiltration of mononuclear leukocytes and some neutrophils. Exudate was minimal or absent. Other lesions included epithelial hypertrophy and hyperplasia, focal epithelial ulceration, and focal to diffuse squamous metaplasia. Goblet cell hyperplasia of mild to moderate severity was observed on the ventral aspect of the nasal septum in rats, but not in mice. Globules of eosinophilic material were observed in the respiratory epithelium of the anterior nasal passages. The globules were present in the basal half of the affected cells, and the dense, oval to round nucleus appeared to be compressed between the globules and the basement membrane. The cell type containing the globules was not identified. Small, basophilic bodies, which were laminated and presumably mineralized, with the globules in mice.
At the 50-ppm exposure level, lesions in the respiratory epithelium were minimal in both rats and mice. Changes were confined to focal squamous metaplasia on the free margins of the turbinates in mice after 6 months of exposure, and eosinophilic globules with mild inflammation and epithelial hypertrophy and hyperplasia after 12 months. A slightly higher incidence of chronic inflammation was observed in the vestibule and the respiratory epithelium of rats in the 10-ppm exposure group.

(2) LESIONS IN THE OLFACTORY REGION
DMA exposure induced a concentration-dependent destruction of the olfactory epithelium which was most severe in the middle third of the dorsal meatus, with variable involvement of the free margins of the ectoturbinates. The most widespread change, which was found consistently in DMA exposed rats and mice, was degeneration of olfactory sensory cells with variable vacuolation of the olfactory epithelium. Another lesion often observed in areas of the olfactory epithelium was characterized by accumulation of hyaline, eosinophilic material in sustentacular cells, which were often markedly hypertrophic. The eosinophilic material was also present as large globules in the overlying airway, suggesting that it may be a secretory product of the sustentacular cells. Similar material was present in the large submucosal glands at the junction of olfactory and respiratory epithelium. These lesions were almost always accompanied by atrophy of the olfactory nerves in the lamina propria. Bowman’s glands exhibited or focal hyperplasia. In the more severely affected cases, olfactory epithelium was replaced by well-differentiated, ciliated respiratory epithelium. These metaplastic areas of ciliated respiratory epithelium were found, in several cases, to be continuous with ciliated ducts of hypertrophic or hyperplastic Bowman’s glands. There were foci of fusiform cells near the basal layer in rats, but not in mice, in areas of respiratory metaplasia in the dorsal meatus. In the underlying, oedematous connective tissue, the basement membrane appeared thickened and separated from the epithelium. Basal cell hyperplasia was frequently observed in the olfactory epithelium of rats, but was not observed in mice. This lesion was characterized by a zone of polyhedral to fusiform cells lying beneath any remaining sustentacular cells. The basal cells had indistinct cytoplasmic boundaries and dark round nuclei and formed cords, sheets, or acinar arrangements. At the 50-ppm exposure level, lesions were much less severe than at 175 ppm and were confined to loss of sensory cells and olfactory nerves, primarily in the middle third of the dorsal meatus. However, most of the animals exposed to 50 ppm exhibited olfactory epithelial lesions. After 12 months, only a few animals at the l0-ppm exposure level were affected, and lesions were confined to focal degeneration of the olfactory epithelium in the dorsal meatus.

Details on results:

Treatment-induced lesions were confined to the nasal passages, and were very similar in nature and grade between the species and sexes. In mice, there was no apparent progression or increase in severity of the nasal lesions from 6 to 12 months, while in rats, increased exposure time was associated with more extensive involvement of the olfactory area. The lesions were present in two areas of the nose: (1) the respiratory epithelium and underlying tissues adjacent to the vestibule, and (2) the olfactory epithelium in the medial portion of the dorsal meatus with variable involvement of more posterior olfactory areas. There was a distinct concentration-response relationship for the severity and frequency of both lesions. After 12 months of treatment, clear effects were seen at 50 and 175 ppm, whereas at 10 ppm lesions were only seen in few animals.

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Deaths at 175 ppm (= 327 mg/m³) in rats and mice were secondary to severe local effects, but should nevertheless be considered to represent systemic toxicity.

Dose descriptor:

LOAEC

Remarks:

local irritation

Effect level:

10 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects on the respiratory epithelium in some of the rats and mice at 12 months after study initiation. Using a conversion factor (1 ppm = 1.87 mg/m³), 10 ppm equals 18.7 mg/m³

Critical effects observed:

not specified

Lesions in Nasal Passages of Rats and Mice Exposed to DMA for 12 Months
DMA (ppm)		Squamous epithelium (level I)^a	Respiratory epithelium (levels I, II)^b	Olfactory epithelium (levels II, III, IV)^c	Chronic inflammation (levels I, II, III)
0	Rats	—	—	+/-	+
	Mice	—	—	—	—
10	Rats	—	+/-	+	+
	Mice	—	+/-	+	—
50	Rats	—	+	++	++
	Mice	—	+	++	+
175	Rats	+/-	+++	+++	++
	Mice	+/-	+++	+++	+
Notes. — =No abnormality detected. +/- = very slight changes of doubtful significance, + = minimal change, ++ = moderate change, +++ = severe change. ^aFor levels of the nose, see Methods. ^bLesion largely confined to the respiratory epithelium adjacent to the vestibule. ^cLesions especially severe in the dorsal meatus. ^dLesions were somewhat more extensive in rats than mice.

 

Conclusions:

In this study the LOAEC for local effects was 10 ppm dimethylamine (DMA), as evidenced by lesions of the respiratory and olfactory epithelium of rats and mice. The NOAEC for systemic toxicity was 50 ppm DMA in both species. Local irritation is the primary mode of action of all free aliphatic amines, and this finding can be transferred to N,N-dimethylbutylamine for assessment.

Executive summary:

Rats (95 Fischer 344/sex and dose) and mice were exposed to atmospheres containing dimethylamine at 0, 10, 50, 175 ppm (exposure duration 12 months, 5 days/week, 6 hours/day). The concentration was monitored using IR spectrophotometry. Groups of rats (and mice) were sacrificed at 6 and 12 months after study initiation. Examinations included clinical signs body weights, clinical chemistry, haematology, necropsy and histopathology.

After 12 months of exposure, the only effect noted was a dose-dependent irritation of the nasal respiratory and olfactory epithelium, both in rats and mice, without any gender or species related differences. At 10 ppm the effects were minimal but present in several animals. Deaths occurred at 175 ppm in high incidence in male mice (Buckley et al, 1985).

 

The results indicate that the mode of action of the free amine is local irritation/corrosion at the point of contact, followed by inflammatory processes with concomitant alterations of the blood cell counts. Systemic toxicity is a minor issue and there was no indication in this study for any kind of systemic toxicity or target organ other than the airways. This result is in line with observations made with other saturated aliphatic amines. Therefore, the result can be read across to related substances, including N,N-dimethylamine, and can be used for assessment.. In this study the LOAEC for local effects was 10 ppm DMA, the NOAEC for systemic toxicity was 50 ppm DMA.

Reference 2

Endpoint:

chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

It is considered justified to utilise information on this substance in a read across approach since, like the registered substance, it is an aliphatic amine with saturated alkyl groups. Furthermore, dimethylamine is considered to be a potential metabolite of the registered substance.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Deaths at 175 ppm (= 327 mg/m³) in rats and mice were secondary to severe local effects, but should nevertheless be considered to represent systemic toxicity.

Dose descriptor:

LOAEC

Remarks:

local irritation

Effect level:

10 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects on the respiratory epithelium in some of the rats and mice at 12 months after study initiation. Using a conversion factor (1 ppm = 1.87 mg/m³), 10 ppm equal 18.7 mg/m³

Critical effects observed:

not specified

Conclusions:

In this study the LOAEC for local effects was 10 ppm dimethylamine (DMA), as evidenced by lesions of the respiratory and olfactory epithelium of rats and mice. The NOAEC for systemic toxicity was 50 ppm DMA in both species. Local irritation is the primary mode of action of all free aliphatic amines, and this finding can be transferred to N,N-dimethylbutylamine for assessment.

Executive summary:

It is considered justified to utilise information on dimethylamine in a read across approach since, like the registered substance, it is an aliphatic amine with saturated alkyl groups. Furthermore, dimethylamine is considered to be a potential metabolite of the registered substance.

Rats (95 Fischer 344/sex and dose) and mice were exposed to atmospheres containing dimethylamine at 0, 10, 50, 175 ppm (exposure duration 12 months, 5 days/week, 6 hours/day). The concentration was monitored using IR spectrophotometry. Groups of rats (and mice) were sacrificed at 6 and 12 months after study initiation. Examinations included clinical signs body weights, clinical chemistry, haematology, necropsy and histopathology.

After 12 months of exposure, the only effect noted was a dose-dependent irritation of the nasal respiratory and olfactory epithelium, both in rats and mice, without any gender or species related differences. At 10 ppm the effects were minimal but present in several animals. Deaths occurred at 175 ppm in high incidence in male mice (Buckley et al, 1985).

The results indicate that the mode of action of the free amine is local irritation/corrosion at the point of contact, followed by inflammatory processes with concomitant alterations of the blood cell counts. Systemic toxicity is a minor issue and there was no indication in this study for any kind of systemic toxicity or target organ other than the airways. This result is in line with observations made with other saturated aliphatic amines. Therefore, the result can be read across to related substances, including N,N-dimethylamine, and can be used for assessment.. In this study the LOAEC for local effects was 10 ppm DMA, the NOAEC for systemic toxicity was 50 ppm DMA.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

18.7 mg/m³

Study duration:

chronic

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

No data were located for N,N-dimethylbutylamine (DMBA). Therefore, a read across approach from data on n-butanol, n-butylacetate and dimethylamine was undertaken.

It is considered justified to utilise information on dimethylamine in a read across approach since, like the registered substance, it is an aliphatic amine with saturated alkyl groups. Furthermore, dimethylamine is considered to be a potential metabolite of the registered substance.

It is considered justified to utilise information on n-butanol in a read across approach since it is a potential metabolite of the registered substance.

It is considered justified to utilise information on n-butylacetate in a read across approach since its metabolite (n-butanol) is also a potential metabolite of the registered substance.

ORAL

- Sub-Chronic, n-butanol (US EPA, 1986; K1)

Four groups of male and female rats were administered the test material daily by gavage at 0, 30, 125 or 500 mg/kg bw/d for either 6 or 13 weeks. Dosing solutions of butanol in deionised water were used.

30 rats per sex per group were dosed, with a further 10 being sacrificed prior to dosing for determination of clinicopathological baseline levels. Ten male and ten female rats from each group were necropsied on study days 43 to 44 (interim sacrifice) and the remaining animals on study days 92 to 93.

Ataxia and hypoactivity (lasting less than 1 h) were observed 2 to 3 minutes after dosing in both sexes of the high-dose group (500 mg/kg bw/d) during the final 6 weeks of dosing. Such ataxia and hypoactivity are typically seen following high oral doses of alcohols. The rapid induction/remission of these effects and the reported increased incidence after the interim kill may be due to the fact that personnel were able to collect post-dose observations more quickly since fewer animals required dosing.

At the interim clinical pathological evaluation, red blood cell count (RBC), packed cell volume (PCV), and haemoglobin (HGB) averages of the 500 mg/kg/day dose group females were 5 % below control averages. Although these differences were statistically significant, they were small and no differences between the parameters were observed in the males of the interim evaluation or between control and treated groups of either sex at the final evaluation. Therefore, even if the lower red blood cell parameters in the 500 mg/kg/day females were an actual treatment-related effect, it was small and transitory and thus not considered as adverse.

Under the conditions of the study the No Observed Adverse Effect Level was determined to be 125 mg/kg bw/day.

- Chronic, dimethylamine (Darad, 1983; K2)

Male wistar rats were administered nitrite (0.2 %) and dimethylamine (DMA) (0.2 %), either singly or in combination for 9 months in drinking waterand the influence on microsomal lipoperoxidation, enzyme activities of acid phosphatase and cathepsin as lysosomal enzymes as well as the enzyme activity of superoxide dismutase (SOD) were evaluated.

The administration of nitrite or DMA appears to decrease the total activity of both the lysosomal enzymes.

In animals fed both nitrite and DMA, the proportion of the free activity of both the enzymes is increased, in spite of a significant reduction in total activity. In these animals, BHT feeding affords marked protection and the increase in the percentage of free activities is restored to control levels.

Nitrite, both singly and in combination with DMA, significantly enhances the level of the enzyme in the supernatant. DMA alone, however, does not have any effect on SOD (superoxide dismutase) activity.

Likewise, rats administered nitrite and DMA are significantly protected against the increase in the enzyme activity, when their diet is supplemented with BHT (butylated hydroxytoluene).

The increased levels of the cytosolic SOD due to the administration of nitrite or DMA may indicate a higher generation of the superoxide radicals. The oxidation of haemoglobin by nitrite has been reported to involve the superoxide radicals.

INHALATION

- Chronic, dimethylamine (Buckley et al, 1985; K2)

Rats (95 Fischer 344/sex and dose) and mice were exposed to atmospheres containing dimethylamine at 0, 10, 50, 175 ppm (exposure duration 12 months, 5 days/week, 6 hours/day). The concentration was monitored using IR spectrophotometry. Groups of rats (and mice) were sacrificed at 6 and 12 months after study initiation. Examinations included clinical signs body weights, clinical chemistry, haematology, necropsy and histopathology.

After 12 months of exposure, the only effect noted was a dose-dependent irritation of the nasal respiratory and olfactory epithelium, both in rats and mice, without any gender or species related differences. At 10 ppm the effects were minimal but present in several animals. Deaths occurred at 175 ppm in high incidence in male mice.

The results indicate that the mode of action of the free amine is local irritation/corrosion at the point of contact, followed by inflammatory processes with concomitant alterations of the blood cell counts. Systemic toxicity is a minor issue and there was no indication in this study for any kind of systemic toxicity or target organ other than the airways. This result is in line with observations made with other saturated aliphatic amines. Therefore, the result can be read across to related substances, including N,N-dimethylamine, and can be used for assessment.. In this study the LOAEC for local effects was 10 ppm DMA, the NOAEC for systemic toxicity was 50 ppm DMA.

- Chronic, dimethylamine (Gross, 1987; K2)

A follow-up 2 year study with dimethylamine in which male rats were dosed at 175.1 ppm demonstrated that areas which exhibit histologic evidence of respiratory metaplasia display the ciliary activity and mucus flow characteristics associated with areas normally lined by respiratory epithelium.

It is interesting that metaplasia to a respiratory-type epithelium occurs at both extremities of the respiratory tract. However, it remains to be determined whether these responses are protective in nature or whether similar mechanisms are responsible for these changes in the nose and lung.

A number of findings were reported here for acute DMA exposures, which were not apparent in animals exposed for 6 or 12 months (Buckley et al., 1985), including focal degeneration of the squamous epithelium in the nasal vestibule and extensive vacuolation of both the respiratory and olfactory epithelia in the anterior nasal passages. Presumably, despite continued exposure, there was considerable repair in the regions lined by squamous epithelium and resolution of the vacuolation seen in the respiratory and olfactory epithelia, following chronic exposure. In the respiratory epithelial-lined regions of the anterior nasal cavity, acute epithelial degeneration led to squamous metaplasia in chronically exposed animals in both the present study and the study reported by Buckley et al. (1985) presumably as a protective or adaptive response.

It is of interest that despite severe tissue destruction in the anterior nose following a single 6-hr exposure, the nasal lesions exhibited very little evidence of progression, even after 2 years of exposure. These findings indicate a possible regional susceptibility to DMA toxicity or a degree of adaptation by the rat to continued DMA exposure.

- Sub-Chronic, n-butyl acetate (David, 1998; K1)

Neurotoxicity investigations were undertaken following repeated dose exposure of the test material (n-butylacetate) to groups of Sprague-Dawley rats. The rats were exposed (whole body) to test material (vapour exposure) at 500, 1500, 3000 ppm for 6 hours a day, 5 days per week, for 13 weeks.

Under the conditions of the study there was no evidence of cumulative neurotoxicity based on a functional observational battery, motor activity measurements, neurohistopathology, and schedule-controlled operant behaviour endpoints. The No Observed Adverse Effect Concentration was determined to be 500 ppm (ca. 2.35 mg/L) .

- Sub-Chronic, n-butyl acetate (David, 2001; K1)

The repeated dose toxicity of the test material was investigated in a study which was conducted in accordance with the standardised guideline EPA OTS 798.2450, and under GLP conditions.

During the study, groups of Sprague-Dawley rats were exposed (whole body) to test material (vapour exposure) at 500, 1500, 3000 ppm for 6 hours a day, 5 days per week, for 13 weeks.

Under the conditions of the study, the No Observed Adverse Effect Concentration was determined to be 500 ppm (ca. 2.35 mg/L) based on reduced body weight and feed consumption noted for the 1500 and 3000 ppm groups. However, there was no systemic or organ-specific toxicity. Degeneration of the olfactory epithelium at concentrations of 1500 and 3000 ppm was observed in areas of the nasal cavity that have demonstrated carboxylesterase activity (Bogdanffy, 1990: Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach. Environmental Health Perspectives 85, 177–186), but there was no evidence of pulmonary toxicity.

- Sub-Chronic, n-butanol (Korsak, 1994; K4)

Sub chronic effects of butan-1-ol were studied in male rats exposed (6h/d; 5d/wk) to 0.15 or 0.31 mg/L (50 or 100 ppm) for 90 days (12 animals per dose).

During the study, there was no mortality and no evidence of clinical signs of toxicity at either exposure concentration. There were no effects on body weight or on absolute or relative (to body weight) organ weights of 7 organs examined. There was a slight but statistically significant decrease in haemoglobin concentration at both exposure concentrations (this did not appear to be dose-related) and a decrease in red blood cell count in the 100-ppm group. There were no statistically significant changes in serum enzymes or in mixed function oxidase activity, however, lipid peroxidation activity was increased at both exposure concentrations (this did not appear to be treatment-related). The authors reported a decreased performance on the rotarod used to assess neurological effects at both concentrations, which increased as the study progressed. The decrease appeared to be dose-related. There were no apparent changes in sensitivity of thermal response at either concentration.

Under the conditions of this study the LOEC was 50 ppm, 0.15 mg/L air (analytical).

DERMAL

- Short-term, n-butanol (McOmie, 1949; K4)

Butan-1-ol was applied to rabbit skin under occlusive conditions for 12* 5 h within 21 d (Omie et al. 1949). Drying of the skin was reported and on continuous exposure, cracking, furrowing and exfoliation of the epidermis. No systemic effects were noted without information of the scope of survey.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to specific target organ toxicity following repeated exposure.