Registration Dossier

Administrative data

Description of key information

Oral: Munley S.M., 2007: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Rats, gavage (0, 250, 500, 1000 mg methylamine hydrochloride/kg bw/day).

Oral: Sarkar, S. N. and Sastry, M.S. Chronic toxicity of methylamine on oral administration and feed contamination in rats., 1990; rats, gavage (10 mg/kg bw/day) and via diet (100 mg/kg bw/ day), up to 90 days. (Diet is a more relevant application route for humans than gavage).
Inhalation: Sriramachari et al., 1994, Comparative toxicity of methyl isocyanate and its hydrolytic derivatives in rats, rats inhalation or subcutaneous injection; 19 µmole or 5.75 mmol/kg respectively, 4 weeks / up to 10 weeks,

no NOAEC could be identified because of only one dose level tested

Inhalation: Kinney et al, 1990, Inhalation toxicology of Methylamine, rats inhalation, 75 ppm, 250 ppm, 750 ppm, 2 weeks.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Rationale for reliability incl. deficiencies:
other:
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
In this justification, the read-across (bridging) concept is applied, based on the chemical structure of the potential analogues, their toxicokinetic behaviour and other available (eco-)toxicological data. Please refer also to the detailed read-across justification attached in section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The underlying hypothesis for the read-across is that the target and the source substance have similar toxicological properties due to their structural similarity, resemblance to their chemical reactivity, and biotransformation products in toxicological compartments. In other words, there is a clear chemical analogy (“biotransformation to common compound", scenario 1 of the Read Across Assessment Framework (ECHA 2017)).
The solvation of both methylamine and methylammonium chloride in water results in solutions of the methylammonium cation (“common breakdown product", scenario 1 of the Read Across Assessment Framework (ECHA 2017)). The toxicity of the respective counterion (“non-common compound” – Cl-) is in this case negligible; as Cl does not drive toxicity effects in mammalian species because it is one of the main electrolytes and is required in large amounts in living organisms. So any toxicity of the amounts originated from the methylammonium chloride at maximum dose levels that would be used in toxicity studies is not expected.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

source substance:
methylamine hydrochloride (methylammonium chloride)
structural formula: CH5N.ClH
SMILES: [Cl-].C[NH3+]
CAS 593-51-1
purity: not specified

target substance:
methylamine
structural formula: CH5N
SMILES: CN
CAS 74-89-5
purity: ≥ 80 – 100 %

No additional information is available on purity of the source and the target substances. Both substances are normally of high purity, containing only minor amounts of impurities that do not influence the read-across validity.

3. ANALOGUE APPROACH JUSTIFICATION
Read across from the structural analogue to the target substance is based on the high structural similarity of the analogue with the substance of interest and the similarity of their toxicological characteristics.
The target substance MMA (CAS 74-59-5), as well as the source substance MMA-HCl (CAS 593-51-1) belong to the category of the “Aliphatic amines” according to the profiler “US EPA New Chemical Categories” in the OECD QSAR Toolbox v4.1.
The basicity of amines increases with the length of the aliphatic rest due to electron releasing properties of alkyl groups: the higher the pKa value, the weaker the acid, so the stronger the base. Monomethylamine (the primary amine with a central nitrogen atom and 1 methyl group and 2 hydrogen-atoms) is the least basic of the aliphatic (primary) amines. Monomethylammonium chloride (composed also of one methyl group attached to the nitrogen atom, which is regarded as the common structure / functional group) is no longer basic, but neutralised (please also refer to the following paragraphs). Therefore, it has to be kept in mind that MMA-HCL is an example of a worst case read-across according to RAAF, as MMA tested as salt may achieve very high doses because it is not corrosive.
Furthermore, the chemicals are characterized by a common Mode Of Action (MOA) in detail as “narcotic amines” according to Acute aquatic toxicity MOA by OASIS in the OECD QSAR Toolbox v4.1.
Methylamine and methylammonium chloride - as primary aliphatic amines - undergo similar reactions and resemble each other in their physico-chemical properties. The fundamental properties of different amine classes (primary, secondary and tertiary) – basicity and nucleophilicity – are very much the same (Morrison and Boyd, 1987).
Typical reactions of amines are salt formation, alkylation, and conversion into amides and Hofmann elimination from quaternary ammonium salts (Morrison and Boyd, 1987).
As already mentioned above, they are linked by the common functionality of one central nitrogen atom which bears an unshared pair of electrons and tends to share these electrons determining a similar chemical behaviour. This unshared electron pair can accept a proton - forming a substituted ammonium ion. The tendency to share this electron pair underlies the entire chemical behaviour of amines as a group and this was considered as main / basic parameter, which is suitable for read-across in an analogue approach within an/a analogue/category approach.
The dissociation constant of MMA allows the conclusion that virtually all molecules of methylamine - when dissolved in an excess of water - are present as the methylammonium cation. Moreover, the available data of MMA with hydrochloric acid shows clearly that there will be no relevant amounts of the amine available once in contact with the bodies’ fluids. Only the ionic form is the relevant species present. This applies to all relevant exposure routes, i.e. inhalation, dermal, and as in this case oral. So, in consequence, the solvation of both methylamine and methylammonium chloride in water would result in solutions of the methylammonium cation (“common breakdown product"). Therefore, one must only regard the physico-chemical properties of the respective counterion. Methylamine solutions are accompanied by the hydroxyl anion OH-, resulting in alkaline solutions, whereas the chloride anion of the methylammonium chloride solutions is not expected to trigger significant changes in the pH and exhibit any significant toxicological effects. Both anions are naturally and ubiquitous occurring ions and are also to a certain extent required for the maintenance of various body functions.
The corrosive effects of MMA can certainly be explained by the high concentration of hydroxyl anions in MMA solution, which are likely to occur even when the gas gets in contact with skin moisture or other body fluids. MMA (gas) is legally classified as Skin Irrit. 2 and Eye Dam. 1, MMA (aqueous solution) is legally classified as Skin Corr. 1B; MMA-HCl has no legal classification for Skin or Eye irritation/corrosion. There is no data available on skin irritation of MMA. Experimental data showed MMA-HCl to be only transiently mildly irritating to the eyes of rabbits (no C & L is triggered). MMA-HCl is expected to be minor irritating when compared to MMA, because of the pH neutralisation caused by HCl. Consequently, the enhanced absorption of MMA compared to MMA-HCl due to damage of the skin barrier should be regarded when the substances are applied in corrosive concentrations or without pH neutralization.
Besides the influence of HCl on the pH value of an aqueous solution, it does not bear a relevant intrinsic property, allowing one in general to focus on the methylammonium cation. Generally, it should be denoted that very commonly in literature there is no differentiation made between MMA and MMA-HCl.
Primary amines are considered to be hydrolytically stable and both substances have been shown to be readily biodegradable. The log Koc values are both negative and relatively close and support the read-across approach. Furthermore, the results show that the substances do not have a significant potential for persistence (not P not vP) or bioaccumulation in organisms (not B not vB). Moreover, they are similar in their toxicity endpoints (despite the above mentioned missing corrosivity in the case of MMA-HCl).
The similar findings (refer also to the data matrix outlined below) for both substances support the conclusion that the identical molecule will be formed from both substances when applied systemically, and this molecule, i.e. the methylammonium cation, is responsible for their behaviour and the observed effects. In consequence, the methylammonium cation is indeed what is left to be considered in both cases and similar effects can reasonably be expected when using data from MMA-HCl for the lacking endpoints, compared to the data obtained with MMA.
Hence, MMA-HCl may perfectly serve as a worst case read-across substance for MMA. So, the available data on MMA-HCl can be used to cover all systemic endpoints currently lacking from MMA, making further testing obsolete.

4. DATA MATRIX
There is data available on the environmental fate and behaviour, ecotoxicological and toxicological properties of MMA. Data on MMA-HCl covers data on Biodegradation, Toxicity to aquatic algae and cyanobacteria, Toxicokinetics, oral Repeated dose toxicity, Genetic toxicity in vivo and Toxicity to reproduction/Developmental toxicity. Hence, the identification and discussion of common properties of MMA and MMA-HCl will be mainly based on this and physicochemical data.
The different physical state of the two substances (MMA is - as a pure substance - gaseous at room temperature, MMA-HCl is a solid primary ammonium salt) triggers some differences in the physico-chemical properties like Melting point, Boiling point, Decomposition temperature and Vapour pressure. Nevertheless, regarding the application of both substances, i.e. their distributed form, the gaseous character of MMA becomes less relevant as the substances are usually not applied in their pure forms but rather as aqueous solutions.
The available data for the following physico-chemical properties, which are among others relevant for absorption, are very similar. Both substances are small molecules with a low molecular weight of 31.042 (MMA) resp. 67.019 (MMA-HCl), they are both very soluble in water (completely miscible in water (MMA) and at least 1080 mg/L at 20°C (MMA-HCl)), have a negative logPow (-0.713 (aqueous solution, 25°C, pH 11.1 - 11.4; MMA) and -3.82 (MMA-HCl)), and both are readily biodegradable. Although being expected to be hydrolytically stable in the natural environment, they both have a very low potential for bioaccumulation in aquatic and terrestrial organisms. Most importantly, MMA has a pKa of 10.79 at 20°C (≙ pKb = 3.21), which indicates that methylamine exists almost entirely in the cationic form as methylammonium cation at pH values of 5 to 9.
MMA (not neutralised) has a toxicity potential towards fish and aquatic invertebrates, but does not have to be classified as hazardous. MMA (neutralised) and MMA-HCl have a lower toxicity potential to aquatic organisms and do not have to be considered to be acutely harmful to fish or aquatic invertebrates, nor to microorganisms (no C & L). MMA and MMA-HCL have been shown to be toxic to aquatic algae and cyanobacteria (shown in Pseudokirchnerella subcapitata, Scenedesmus obliquus and Microcystis aeruginosa). Their aquatic toxicity potential under real environmental conditions has to be judged carefully, since, methylamine and methylamine hydrochloride are readily biodegradable in nature. As such, both can be considered as non-toxic to aquatic organisms and thus do not have to be classified as hazardous as per the CLP classification criteria.
Regarding their toxicity towards mammals, both substances exert their acute toxicity oral toxicity in the same range and both are classified as Acute tox 4 and MMA also as STOT SE 3 (C≥5 %). Moreover, MMA is corrosive / irritative to the skin and the eyes (MMA (gas) = Skin Irrit. 2 and Eye Dam 1; MMA (aqueous solution) = Skin Irrit 1B); in the case of MMA –HCl no classification is warranted.
Regarding their repeated dose toxicity, the No-observed-adverse-effect-levels of both substances to differ to a certain extent (NOAEL ≥ 10 versus NOAEL = 500 mg/kg bw/day). For MMA HCL there is also data for toxicity to reproduction and developmental toxicity available (NOEL (reproduction/systemic tox) = 500 mg/kg bw/day and NOAEL (maternal/developmental tox.) = 155 mg/kg bw/day).
No data on long-term toxicity to fish; on long-term toxicity to aquatic invertebrates or carcinogenicity were available.
Reason / purpose:
read-across source
No. of animals per sex per dose:
12/sex/dose
Clinical signs:
no effects observed
Description (incidence and severity):
No specific test substance-related deaths in males or females at any level tested, no test substance-related clinical observations at any level tested
Mortality:
no mortality observed
Description (incidence):
No specific test substance-related deaths in males or females at any level tested, no test substance-related clinical observations at any level tested
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, adverse effects at 1000 mg/kg/day: significant reductions in body weight in P1 animals. No test substance-related effects on body weight parameters at 500 mg/kg/day or lower.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, adverse effects at 1000 mg/kg/day: slightly significant reduction in food consumption parameters in P1 animals. Did not persist longer than 2 weeks. No effects on maternal food consumption during gestation at 500 mg/kg/day or lower
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Compound-related but not adverse(females), unrelated effects (males) changes in haematologic parameters
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No adverse changes in clinical chemistry. minimal changes in Cholesterol, Glucose, Globulin, total protein, inorganic phosphorus, AP activity, tryglycerides (dose ranges: 500 - 1000 mg/kg /day). Changes mainly likely to be related but not adverse
Urinalysis findings:
no effects observed
Description (incidence and severity):
No adverse changes in urinalysis parameters, minimal changes in urine osmolarity, pH, ketones (dose ranges: 500 - 1000 mg/kg /day). Changes mainly likely to be related but not adverse
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test substance-related effects or statistically significant differences on fore- / hinglimb grip strength. No test substance-related effects or statistically significant effects for any behavioral parameter. No effects on duration of movement.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver and kidney weight parameters were increased in both sexes. Liver weights were increased in males at ≥ 250 mg/kg/day and in females at ≥ 500 mg/kg/day. Kidney weights were increased in both sexes only at the highest dose (1000 mg/kg/day).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, adverse effects at 1000 mg/kg/day: significantly reduced corpora lutea counts and subsequently lower implantation site counts and litter sizes.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings were observed in the trachea (squamous metaplasia) and stomach (mucoid metaplasia) of males and females given 1000 mg/kg/day of the test substance.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
There was a test substance-related and statistically significant reduction in mean corpora lutea count at 1000 mg/kg/day that resulted in a reduction in the mean number of implantation sites per litter and a subsequent significant reduction in the mean litter size at this dose level. There were no other test substance-related effects on reproductive outcome at this level nor were there any effects on reproductive outcome at 500 mg/kg/day or lower.
There was a significant reduction in mean litter size at 1000 mg/kg/day that was associated with a test substance-related reduction in the mean numbers of corpora lutea observed at this level. Otherwise, there were no additional test substance-related effects in offspring at 1000 mg/kg/day and no evidence of any test substance-related effects on offspring at 500 mg/kg/day or lower.

There were no test substance-related clinical observations in the pups at any dose level tested.

There were no test substance-related effects on mean pup weight either at birth or on day 4 of lactation at any dose level tested.
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw (total dose)
Based on:
other: reductions in parental body weights and food consumption and effects on reproductive outcome (reduced corpora lutea and subsequent reductions in implantations and litter size).
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: Reproductive outcome
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Pathology
Critical effects observed:
not specified

A. Test Substance Stability (Appendix B): Alfa Aesar supplied Haskell Laboratory with the test substance Methylammonium chloride, conducted the purity analysis for the beginning of the study, and supplied the Certificate of Analysis (COA) confirming the purity of the test substance Methylammonium chloride to be 99.46%. The stability of the test substance methylamine hydrochloride was confirmed by purity analysis conducted near the end of the study by Case Consulting Laboratories, Inc., confirming the purity of the test substance methylammonium chloride to be 99.2% (average of 2 samples analyzed).

B. Test Formulation Analysis (Appendix C): The analytical method used is described in Appendix C. Data from the analysis of the samples during the study indicate that the test substance Methylammonium chloride was at the targeted levels, mixed uniformly, and was stable under the conditions of the study. No test substance was found in the 0 mg/mL samples.

1. Stability of the Test Substance in the Vehicle: The data show that the concentrations of the test substance Methylammonium chloride in the vehicle were stable for the 7 days of refrigeration followed by 5 hours at room temperature.

2. Homogeneity and Concentration Verification: The data for samples submitted show that the concentrations of the samples were at the targeted levels and were uniformly mixed.

Conclusions:
Test substance-related effects were observed at 1000 mg/kg/day and were limited to reductions in parental body weights and food consumption parameters and a significant reduction in the mean corpora lutea and associated reduction in mean implantation sites and mean litter size. There was no evidence of systemic or reproductive toxicity at 500 mg/kg/day or lower.
Executive summary:

Test substance-related effects were observed at 1000 mg/kg/day and were limited to reductions in parental body weights and food consumption parameters and a significant reduction in the mean corpora lutea and associated reduction in mean implantation sites and mean litter size. There was no evidence of systemic or reproductive toxicity at 500 mg/kg/day or lower. Under the conditions of the study, there were no test substance-related effects on any neurobehavioral parameter evaluated in either males or females at any dose level tested. Daily gavage administration of up to 1000 mg/kg/day of the test substance Methylammonium chloride to male and female rats resulted in no adverse changes in clinical pathology parameters.

Liver: Since there was no gross or microscopic finding that correlated with the increased liver weights, and there was no increase in serum liver enzymes (see Clinical Chemistry), the weight effect was considered pharmacological and not adverse. Exposure to xenobiotics commonly induces hepatic metabolic enzymes in laboratory animals. While microscopic hypertrophy of hepatocytes may occur in hepatic enzyme induction, small increases in liver weight parameters are often not associated with a morphological change.

The increased kidney weight parameters in both sexes at 1000 mg/kg/day did not correlate with any gross or microscopic renal pathology. As in liver hepatocytes, xenobiotics may induce P450 microsomal enzymes in the renal proximal tubular epithelium. Increased kidney weights often precede morphological changes (i.e., hypertrophy) in non-adverse enzyme induction.

The metaplasia consisted of the partial replacement of the normal ciliated pseudostratified columnar epithelium on the ventral aspect of the tracheal mucosa with round and slightly flattened epithelial cells resembling mucous membrane. The change was not associated with inflammation of either the trachea or the lung. The minimal to mild squamous metaplasia was probably the result of transient topical exposure of the tracheal mucosa to the test substance Methylammonium chloride following gavage administration. It was interpreted to be a non-adverse adaptive response, most likely reversible, that was due to imperfect dosing or transient esophageal reflux.

The metaplasia consisted of a partial replacement of the normal parietal cells in the gastric glands with mucous epithelial cells. The change was evident in the fundic region of the glandular mucosa adjacent to the esophageal inlet. The change was not associated with any inflammation and was considered to be non-adverse. The minimal mucoid metaplasia was probably the result of the daily gavage administration of a slightly irritating solution to the glandular mucosa adjacent to the esophageal inlet. The degree of focal metaplasia appeared to be biologically insignificant. As with the focal tracheal metaplasia, the focal gastric metaplasia was interpreted to be a non-adverse, most likely reversible, adaptive response.

---

Daily gavage administration of 0, 250, 500, and 1000 mg/kg/day of the test substance methylamine hydrochloride, to male and female rats for 98 to 119 consecutive days, resulted in increased liver and kidney weights, squamous metaplasia of the tracheal mucosa, and mucoid metaplasia of the glandular gastric mucosa. The increased liver weights in males (≥ 250 mg/kg/day) and females (≥ 500 mg/kg/day) and kidney weights in both sexes (1000 mg/kg/day) were consistent with pharmacological enzyme induction and were considered to be non-adverse. Minimal to mild squamous metaplasia of the tracheal mucosa and minimal focal mucoid metaplasia of the gastric glandular mucosa of several high-dose rats of both sexes were interpreted to be the result of topical exposure of the test substance Methylammonium chloride. The metaplastic changes in both tissues were interpreted to be adaptive and probably reversible. Therefore, both the tracheal and gastric metaplasia were considered to be non-adverse. There were no test substance-related effects on cause of death, gross pathology, or reproductive failure in adult rats. Gross examination of nursing pups that died before day 4 of lactation did not reveal any test substance-related findings. Under the conditions of this study, the no-observed-effect level (NOEL) for pathology for male and female rats was the highest dose tested, 1000 mg/kg/day.

Under the conditions of this study, the no-observed-effect level (NOEL) for systemic and reproductive toxicity was 500 mg/kg/day Methylammonium chloride based on reductions in parental body weights and food consumption and effects on reproductive outcome (reduced corpora lutea and subsequent reductions in implantations and litter size).

The target substance methylamine and the source substance methylammonium chloride used in this study belong to the group of primary aliphatic amines. The solvation of both, methylamine and methylammonium chloride in water results in solutions of the methylammonium cation (common "breakdown product"). Both respective counterions are naturally and ubiquitous occurring ions and are also to a certain extent required for the maintenance of various body functions. Besides the influence on the pH value of an aqueous solution (OH-), they do not bear a relevant intrinsic property, allowing one in general to focus on the methylammonium cation. The methylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms.

Therefore both substances are expected to follow the same toxicokinetic pattern.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported, published 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Acceptable, well-documented publication which meets basic scientific principles. 1. brief documentation and description, publication is a mix of several studies 2. evaporation only of the diet dosing can not bei completly excluded, but minimizing strategies are undertaken
Reason / purpose:
reference to other study
Qualifier:
no guideline required
Principles of method if other than guideline:
no data available because of publication
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Route of administration:
other: gavage and via diet
Vehicle:
not specified
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
21, 45, 65 and 90 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations: 10 mg/kg bw
Basis: other: by gavage
Remarks:
Doses / Concentrations: 100 mg/kg bw
Basis: nominal in diet
No. of animals per sex per dose:
10 animals per dose.
Control animals:
yes, concurrent no treatment
Details on study design:
- Groups of 10 weaned male animals received 10 mg/kg bw by gavage or 100 mg/kg bw diet for 21, 45, 65, and 90 days
- Body weights (weekly), liver, heart, lungs, kidneys and adrenals (weight and macroscopic finding) were recorded; ALT, AST, LDH and alkaline phosphatase activities were measured
Observations and examinations performed and frequency:
Body weights (weekly)
Sacrifice and pathology:
liver, heart, lungs, kidneys and adrenals (weight and macroscopic finding) were recorded
Other examinations:
ALT, AST, LDH and alkaline phosphatase activities were measured
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:


BODY WEIGHT AND WEIGHT GAIN
Growth rate study for 90 days in both the groups treatedwith MA either orally or incorporated in the diet show ed linear growth and no significant difference in the body weights was observed at the end 90 days compared to those of the controls. Body weights at 21, 45 and 65 days were also not significantly different in any of the groups.




CLINICAL CHEMISTRY
Enzymathic activities (units/litre) of serum GOT (46±4.27), GPT (16.5 ± 1.0), LDH (506 ± 28.5) and alkaline phosphatase (104.2 ± 10.6) in the control group were not significantly different from the treated groups irrespective of duration of trials ranging from 21 to 90 days, indictaing that the levels of methylamine used had no adverse effect on the functions of liver, heart and other vital organs since any alteration in enzyme activity is an index of pathological process and the target organs.

ORGAN WEIGHTS
organ weights of liver, heart, lungs, kidneys, testes and adrenal glands calculated per 100 g body weight in the control group were not significantly different from the tretated groups at the end of 90 days. Similar findings were recorded in respect of trials for 21, 45 and 65 days.

GROSS PATHOLOGY

Toxicity of a chemical is normally reflected in the target organs and the absence of any difference in the organ weights or gross lesions in the treated groups indicated the nontoxic character on the vital organs at the dose levels ingested and used in the present studies.


Distribution of MA in tissues:
There were no residues of MA found in the meat of rats, which could be due to detoxification and high water solubility of the chemical
Dose descriptor:
other: LD50
Effect level:
375 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: in weaned rats (preliminary study)
Dose descriptor:
other: LD50
Effect level:
80 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: in adult rats (preliminary study)
Dose descriptor:
NOAEL
Remarks:
(by gavage)
Effect level:
>= 10 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: no effects
Dose descriptor:
NOAEL
Remarks:
in diet
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: no effects
Critical effects observed:
not specified
Conclusions:
Methylamine did not cause adverse effects if administered both by gavage or in diet to rats during 90 days. No treatment related effects
Body weight gains, organs weights & enzyme activities similar to controls.
Executive summary:

Effect of methylamine on growth rate, organ weights, activities of some serum enzymes, accumulation of residues in the tissues and reproductive performance have been carried out in the rats to evaluate short- and long-term residual effects on oral ingestion. The test material was administered to the rats by gavage (10 mg/kg bw) daily and in diet (100 mg/kg bw). The rats received the test material during 21, 45, 65 and 90 days.

No treatment related effects were recorded in animals both if administered by gavage or in diet. Body weight gains, organs weights and enzym activities tested were similar to those of controls.

Endpoint conclusion
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
repeated dose toxicity: inhalation
Remarks:
other: subacute and chronic
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable well-documented publication, which meets basic scientific principles
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Long-term (subacute and chronic) histopathological effects in the lungs of rats subjected to a single exposure to methyl isocyanate (MIC) by both the inhalation and subcutaneous (s.c.) routes as well as the role of methylamine (MA) and N,N'-dimethylurea (DMU), the hydrolytic derivatives of MIC in eliciting the observed changes.
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
no data
Route of administration:
other: inhalation and subcutaneous application
Type of inhalation exposure:
not specified
Vehicle:
not specified
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
For MA by the inhalation route for each period of study, four rats were exposed to 19 µmol/L (~1.0 LC50 MIC in terms of mol).
In addition, either MA or DMU at 5.75 mmol/kg was injected s.c. into a group of four rats each for each period of the study.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
no data
Frequency of treatment:
no data
Remarks:
Doses / Concentrations: Inhalation: 19 µmol/L
Basis: nominal conc.
Remarks:
Doses / Concentrations: s.c. : 5.75 mmol/L
Basis:
No. of animals per sex per dose:
4
Control animals:
yes
Details on study design:
In the corresponding control groups for each duration of study, four rats each were subjected to either the inhalation procedure without the test material or s.c. administration of the vehicle, olive oil.
Observations and examinations performed and frequency:
The appearance and behavior of animals exposed to MIC and its hydrolytic derivatives have already been described in the preceding paper (Jeevaratnam and Sriramachari 1994). Nearly similar findings were found in all the rats after the exposure to varied concentrations of MIC. The animals continued to be moribund and scarcely consuming any feed or water. After 2 or 3 days, the general condition of the animals improved and they were moving about, Although many of them exhibited the signs and symptoms of acute respiratory distress syndrome (ARDS) up to 4 weeks, there was a gradual improvement in ARDS beyond 4 weeks. Thereafter, the animals were apparently normal without any signs of underlying gross pathology of the lung.
Sacrifice and pathology:
Only a group of four rats of those that survived were killed at the end of each duration of the experiment by cervical dislocation. They were subjected to post-mortem examination.
Other examinations:
Although all the viscera were removed at autopsy and fixed in 10% neutral formalin, only the histopathological changes of the lungs are dealt in this communication. The tissues wereb processed through paraffin and stained by H&E, PAS, reticulin (silver) and collagen (tfichrome) using standard procedures.
Statistics:
no data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see in examinations
Mortality:
mortality observed, treatment-related
Description (incidence):
see in examinations
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
bronchial lumen and the epithelial lining were apparently normal in both the MA groups, peculiar and extensive edema in rats exposed to MA via inhalation
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see in details
Details on results:
Methylamine: at all periods of the study, the bronchial lumen and the epithelial lining were apparently normal in both the MA groups.
A peculiar and extensive edema was observed at the end of 1 week in rats exposed to MA vapors through inhalation route. The lungs were uniformly pale and had a washed out appearance. Histologically, lung parenchyma was filled with transparent clear aqueous fluid with the widespread separation of tissue spaces. Thus, the mucosal lining was lifted off the submucosa. Similarly, the perivascular areas were filled with clear edematous fluid extending throughout the pulmonary interstitium and alveoli. This phenomenon was seen to extend into the walls of blood vessels whose adventitial muscular coats were widely separated (Fig. 10). The faintly PAS positive reaction indicated that the fluid is an exudate rather than a transudate.

After 1 week: In both the MA groups, the interstitial spaces showed varying degrees of infiltrates characterized by mononuclear cells and histiocytes without any foamy cells. The connective tissue fibers were sparse and inconspicuous as revealed by both the silver and trichrome preparation.

After 4 weeks: A moderate to severe interstitial pneumonitis without edema was noticed in lungs of rats exposed to MA by either route, the severity being more pronounced in the subcutaneous route of exposure. The reticulin staining showed moderately severe fibrillogenesis in the highly cellular interstitial septa.

After 10 weeks: In both the groups, the lung showed severe interstitial pneumonitis extending into peribronchial and perivascular areas (Fig. 12a). The silver preparation showed abundance of collagen fibers in the corresponding areas (Fig. 12b). By comparison, the intensity of the above lesions is less in MA treated rats than in the MIC treated rats.
Dose descriptor:
NOAEC
Basis for effect level:
other: one dose level tested; severe interstitial pneumonitis
Remarks on result:
not determinable
Remarks:
no NOAEC identified
Critical effects observed:
not specified
Conclusions:
MA causes pumonary edema 1 week after expose to MA by inhalation.
MA causes interstitial pneumonitis progressing to fibrosis.
Executive summary:

Sriramachari et al. performed in 1994 a subacute and chronic test in rats with monomethylamine by the route of inhalation or subcutaneous administration. The test duration was 4 weeks; the substance was administered at concentrations of 19 µmole or 5.75 mmol/kg respectively. The animals showed a treatment-induced accumulation of hemorrhagic fluid in the pleural cavity. Histological examination revealed a severe interstitial pneumonitis and fibrosis.

At the subacute phase, the intraalveolar and interstitial edema were prominent only in the inhalation group as against the more pronounced inflammatory response in the s.c. route. With the progress of time the evolution of lesions appeared to be similar, culminating in the development of significant interstitial pneumonitis and fibrosis. MA, one of the hydrolytic derivatives of MIC, also caused interstitial pneumonitis progressing to fibrosis, albeit to a lesser extent than MIC, indicating its contribution to the long-term pulmonary damage.

Though the mechanisms by which either MIC or MA elicit the observed long-term effects are not known presently, the fact that the lung is the target organ for both MIC and MA, which cause the progressing changes leading to a unified picture of chronic puhnonary fibrosis, merits further consideration.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable well-documented publication, which meets basic scientific principles
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Principles of method if other than guideline:
to determine the effect of MA following repeated inhalation exposures, they studied the response of the rat following a series of 10 subchronic exposures.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD) BR
Sex:
male
Details on test animals and environmental conditions:
Young adult male Crl:CD(SD) BR rats were received from Charles River Breeding Laboratory, Kingston, N.Y. and quarantined for 1 wk prior to testing. During the test, rats were housed in pairs in 8 x 14 x 8 in. suspended, steel-mesh cages. Animal rooms were maintained at 50 +/- 10 % relative humidity and 23 +/- 2 °C on a timer-controlled 12/12 lightdark cycle. Except during exposure, Purina Certified Rodent Chow 5002 and water were available ad libitum.
Route of administration:
inhalation: gas
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Four groups of 10 male rats, each 8 wk old and weighing between 234 and 251 g, were placed in cylindrical stainless-stelle holders equipped with coinical nose pieces. The restrainers were inserted into face plates on the exposure chambers such that the nose of each rat protruded into the chamber. The fourth group was a procedural conctrol group that was exposed to air only (no MA).
Gaseous atmospheres of MA were generated by dilution of pure MA with air. Polyethylene gas sample bags were filled with MA, and the gases were subsequently metered into three-neck mixing flasks with FMI (Fluid Metering, Inc., Oyster Bay, N.Y.) pumps. The pumps were chosen with capacities sufficient to produce the design concentrations of 75, 225 and 750 ppm when mixed with 15 L/min of dilution air. The diluted gases were directed into siliconized, 20-L-cylindrical glass exposure chambers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentrations were determined at 30 min intervals during the exposures. chamber atmospheres were pumped continously into Miran model 1A infrared spectrometers at a flowrate of 5 L/min. Analysis for MA was conducted at a wavelength of 3,41-3,42 µm, the absorbance maximum for C-H bond streching. the infrared spectrometers were operated with a 1 mm slit width and path lengths of 6.75 m (high leve) and 20.25 m (low and intermediate levels). Chamber concentrations were determined by comparison to freshly prepared MA standards.
Duration of treatment / exposure:
6 h per day
Frequency of treatment:
5 exposure days, 2 rest days, 5 exposure days
Dose / conc.:
75 ppm (nominal)
Dose / conc.:
225 ppm (nominal)
Dose / conc.:
750 ppm (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
All rats were weighted and observed for clinical signs daily (excluding the 2-d rest period) and for the 14-d postexposure recovery period.
Sacrifice and pathology:
Immediately following the tenth exposure, 5 rats were readied for necropsy, the remaining 5 rats/group were retained for a 14-d nonexposure recovery period.
Clincal laboratory examinations were performed in 10 rats/group following 10 exposures and on 5 rats/group following the 14-d recovery period.
With the exception of the 750 ppm exposure group, 5 rats per group were prepared for necropsy after the tenth exposure and the remaining rats were prepared for necropsy on the forteenth recovery day for gross and histopathologic examination. In the 750 ppm group, tissues from rats dying from exposures 8-10 were examined, and 3 rats were studied after exposure 10 and 2 were sacrificed after the recovery period, using chloroform anesthesia and exsanguination. Organs and tissues examined included the heart, lungs, nose, trachea, liver, pancreas, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, kidneys, urinary bladder, bone marrow (sternal), spleen, thymus, mediastinal lymph nodes, thyroid, testes, epididymides, adrenal glands, brain, skin, nose, and eyes. All other gross lesion including tissue masses wer also examined. Tissues were fixed in either 10 % neutral buffered formalin of Bouin's solution and routinely processed for light microscopy.
Organ weights and organ-to-body ratios were measured or calculated for the heart, lungs, liver spleen, kidneys, testes and thymus.
Other examinations:
Indivitual urine samples were collected from rats housed overnight in stainless-steel metabolism cages. Samples were analyzed for voloume, osmalarity, pH, blood, sugar, protein, bilirubin, urobilinogen, and ketone. Each specimen was noted for color and appearance, and the sediment from pooled specimens was examined microscopically.
Blood samples were taken from the tail vein following collection of the urine sample. Whole blood samples were analyzed for erythrocyte count, haemoglobin concentration, mean corpuscular volume, platelet count, leukocyte count, and realtive numbers of neutrophils, band neutrophils, lymphcytes, atypical lymphocytes, eosinophils, monocytes and basophils. Haematocrit, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentrations were calculated from the erythrocyte data. Serum samples were anlyzed for alkaline phosphatase, alanine aminotransferase, and aspartat aminotransferase activities, and urea nitrogen, creatinine, total protein, and cholesterol concentrations
Statistics:
For statistical analyses, a one-way analysis of variance (ANOVA) and Bartlett's test were used at each sampling time. When the F-test from ANOVA was significant, the Dunnett test was used to compare means from the control group with each of the groups exposed to MA. test groups were compared with the appropriate control group by least significant difference and Dunnett's test when the ratio of vaiance (F) indicated significant amoug-to-within group variatins. Significance was judged at the .05 probability level.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
rats exposed to 750 ppm: hyperactivity, aggresion, hunched posture, lung noise, labored brathing, gasping, dry red nasal and ocular discharges, salivation, pallor, wet perineum, diarrhea, ruffled fur and facial hair loss.
Mortality:
mortality observed, treatment-related
Description (incidence):
rats exposed to 750 ppm: hyperactivity, aggresion, hunched posture, lung noise, labored brathing, gasping, dry red nasal and ocular discharges, salivation, pallor, wet perineum, diarrhea, ruffled fur and facial hair loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights for rats exposed to 750 ppm were sig. lower than the controls beginning on the third exposure day and persisting through the fourteenth day of recovery. No compound related changes were seen in rats exposed to 75 / 250 ppm.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative weights for a number of organs were signficantly lower in rats exposed to 75 ppm, but when these weights were adjusted for body weight, these differences became less although they remained statistically significant.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
After the tenth exposure, rats in the 750 ppm group hat increased erythrocyte, neutrophil, and monocyte counts, haemoglobin concentrations and haematocrit. increases in serum alanine and aspartate aminotransferase activities, as well as blood urea nitrogen
Details on results:
At the 750 ppm exposure levels, 4 rats died or were killed in extremis by the tenth exposure day. One rat also died after 12 d of recovery. All of these rats, together with 3 rats that were sacrificed following the tenth exposure, had similar gross and microscopic findings. These changes consisted of a distended gastrointestinal tract, small spleen and thymus due to depletion of lymphocytes, bone marrow hypocellularity, focal hepatic necrosis, dark of dull red lungs, and red nasal discharge. Nasal degeneration, severe necrosis leading to atrophy, and septal perforation were seen in the nasal turbinate mucosa.
In the 250 ppm exposure group, all rats examined after the tenth exposure had erosions and/or ulcerations of the nasal turbinate mucosa. No other lesions were seen in rats exposed to 250 ppm MA, and only mild nasal turbinate irritation was seen in rats expose to 75 ppm MA.
Dose descriptor:
LOAEC
Effect level:
75 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: local irritation in respiratory tract
Remarks on result:
other: corresponds to 96 mg/m3
Dose descriptor:
LOAEC
Effect level:
250 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: corresponds to 322 mg/m3
Critical effects observed:
not specified

Four rats in the 750 ppm group were found dead of were sacrificed in extermis on the eighth of tenth exposure day.

Although a red nasal discharge was seen in all groups, its severity was greater among rats exposed to 250 or 750 ppm.

No other signs of response to MA were seen in rats exposed to 250 ppm, and no unusual outward signs were seen in rats exposed to 75 ppm. A clear nasal discharge was also observed in rats from all treatment groups (including the controls) with equal severity and incidence.

During the 14 -d recovery period, lung noise, clear or dry red nasal discharges, ocular opacity, pallor, diarrhea, and discolored fur were observed in rats exposed to 750 ppm.

One additional rat from the 750 ppm group died on the twelfth day of the recovery period. No abnormal clinical signs were seen in any of the other groups of rats during the recovery period.

Conclusions:
It is concluded that MA can produce changes in the upper respiratory tract of the rat following repeated exposure to 250 ppm. Higher concentrations were not well tolerated and led to death, liver damage, and changes in the hematopoetic system. Exposure to 75 ppm approached the no-observed-adverse-effect level under these test conditions. Based on the results of this study, 75 ppm is the LOAEC for local effects in the respiratory tract, whereas 250 ppm is the LOAEC for systemic effects. The durrent TLV of 10 ppm for MA, based on analogy to ethylamine (ACGIH, 1986), appears to be appropriate and protective.
Executive summary:

In a study performed by Kinney et al. in 1990, methylamine produced only a mild irritation of the nasal turbinate mucosa in rats when exposed to 75 ppm for 10 days. Exposure to 250 ppm produced damage, restricted to the respiratory mucosa of the nasal turbinates of the rats (focal erosion and/or ulceration). Exposure to 750 ppm for 10 days led to severe body weight losses, clinical pathologic changes suggestive of liver damage, nasal degenerative changes, and haematopoietic changes, not all of which were reversible during the recovery period. Repeated exposure to 250 ppm MA produced focal erosion and/or ulceration of the nasal turbinate mucosa after the tenth exposure. This suggests that the material is absorbed in the anterior portion of the upper respiratory tract. No signs of focal or diffuse irritation were seen further into the respiratory tract, including the trachea and the lungs. All clinical chemical, haematologic, and anatomic pathologic findings, with the exception of the elevated blood urea nitrogen values, were normal after the recovery period. It thus appears that 250 ppm MA is relatively well tolerated by rats following 10 exposures and that the target tissue is the respiratory mucosa of the nasal turbinates. The lesion produced by this exposure regimen is mild but not reversible. Repeated exposure to 750 ppm was not well tolerated by rats. A number of mortalities occurred late in the exposure period, and one rat died late in the recovery period. An immediate response was reflected by weight loss, which persisted throughout the recovery period. Severe distention of the gastrointestinal tract, discolored lungs, nasal degeneration, and red nasal discharge were the predominant signs in rats either found dead, killed in extremis, or sacrificed after the tenth exposure. Lymphoid depletion in the thymus, spleen, and bone marrow is interpreted as being a secondary effect reflecting the stress of the exposure (severe weight loss) rather than a specific response to MA. The increased serum cholesterol concentration and alanine and aspartate aminotransferase activities and decreased serum protein concentration reflect the effect of MA on both the integrity (enzyme alterations) and function (protein, cholesterol) of the hepatic tissues. The increased red cell count, haematocrit, and haemoglobin concentration probably reflect a mild treatment-related absolute polycythaemia. This is consistent with relative polycythaemia (haemoconcentration), which may explain the increased BUN immediately following exposure and at the end of the recovery period. The increased numbers of neutrophils and monocytes and the increased acidity of the urine after the tenth exposure appear to be related to MA exposure. It is concluded that MA can produce changes in the upper respiratory tract of the rat following repeated exposure to 250 ppm. Higher concentrations were not well tolerated and led to death, liver damage, and changes in the haematopoetic system. Exposure to 75 ppm approached the no-observed-adverse-effect level under these test conditions. Based on the results of this study, 75 ppm is the LOAEC for local effects in the respiratory tract, whereas 250 ppm is the LOAEC for systemic effects. The current TLV of 10 ppm for MA, based on analogy to ethylamine (ACGIH, 1986), appears to be appropriate and protective.

For calculation of the derived no effect levels a LOAEL of 75 ppm (96 mg/m³) for local effects and a LOAEL of 250 ppm (322 mg/m³) for systemic effects, extracted out of the above mentionned clinical findings is used further for the risk characterization

Endpoint conclusion
Dose descriptor:
LOAEC
322 mg/m³
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable well-documented publication, which meets basic scientific principles
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Principles of method if other than guideline:
to determine the effect of MA following repeated inhalation exposures, they studied the response of the rat following a series of 10 subchronic exposures.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD) BR
Sex:
male
Details on test animals and environmental conditions:
Young adult male Crl:CD(SD) BR rats were received from Charles River Breeding Laboratory, Kingston, N.Y. and quarantined for 1 wk prior to testing. During the test, rats were housed in pairs in 8 x 14 x 8 in. suspended, steel-mesh cages. Animal rooms were maintained at 50 +/- 10 % relative humidity and 23 +/- 2 °C on a timer-controlled 12/12 lightdark cycle. Except during exposure, Purina Certified Rodent Chow 5002 and water were available ad libitum.
Route of administration:
inhalation: gas
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Four groups of 10 male rats, each 8 wk old and weighing between 234 and 251 g, were placed in cylindrical stainless-stelle holders equipped with coinical nose pieces. The restrainers were inserted into face plates on the exposure chambers such that the nose of each rat protruded into the chamber. The fourth group was a procedural conctrol group that was exposed to air only (no MA).
Gaseous atmospheres of MA were generated by dilution of pure MA with air. Polyethylene gas sample bags were filled with MA, and the gases were subsequently metered into three-neck mixing flasks with FMI (Fluid Metering, Inc., Oyster Bay, N.Y.) pumps. The pumps were chosen with capacities sufficient to produce the design concentrations of 75, 225 and 750 ppm when mixed with 15 L/min of dilution air. The diluted gases were directed into siliconized, 20-L-cylindrical glass exposure chambers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentrations were determined at 30 min intervals during the exposures. chamber atmospheres were pumped continously into Miran model 1A infrared spectrometers at a flowrate of 5 L/min. Analysis for MA was conducted at a wavelength of 3,41-3,42 µm, the absorbance maximum for C-H bond streching. the infrared spectrometers were operated with a 1 mm slit width and path lengths of 6.75 m (high leve) and 20.25 m (low and intermediate levels). Chamber concentrations were determined by comparison to freshly prepared MA standards.
Duration of treatment / exposure:
6 h per day
Frequency of treatment:
5 exposure days, 2 rest days, 5 exposure days
Dose / conc.:
75 ppm (nominal)
Dose / conc.:
225 ppm (nominal)
Dose / conc.:
750 ppm (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
All rats were weighted and observed for clinical signs daily (excluding the 2-d rest period) and for the 14-d postexposure recovery period.
Sacrifice and pathology:
Immediately following the tenth exposure, 5 rats were readied for necropsy, the remaining 5 rats/group were retained for a 14-d nonexposure recovery period.
Clincal laboratory examinations were performed in 10 rats/group following 10 exposures and on 5 rats/group following the 14-d recovery period.
With the exception of the 750 ppm exposure group, 5 rats per group were prepared for necropsy after the tenth exposure and the remaining rats were prepared for necropsy on the forteenth recovery day for gross and histopathologic examination. In the 750 ppm group, tissues from rats dying from exposures 8-10 were examined, and 3 rats were studied after exposure 10 and 2 were sacrificed after the recovery period, using chloroform anesthesia and exsanguination. Organs and tissues examined included the heart, lungs, nose, trachea, liver, pancreas, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, kidneys, urinary bladder, bone marrow (sternal), spleen, thymus, mediastinal lymph nodes, thyroid, testes, epididymides, adrenal glands, brain, skin, nose, and eyes. All other gross lesion including tissue masses wer also examined. Tissues were fixed in either 10 % neutral buffered formalin of Bouin's solution and routinely processed for light microscopy.
Organ weights and organ-to-body ratios were measured or calculated for the heart, lungs, liver spleen, kidneys, testes and thymus.
Other examinations:
Indivitual urine samples were collected from rats housed overnight in stainless-steel metabolism cages. Samples were analyzed for voloume, osmalarity, pH, blood, sugar, protein, bilirubin, urobilinogen, and ketone. Each specimen was noted for color and appearance, and the sediment from pooled specimens was examined microscopically.
Blood samples were taken from the tail vein following collection of the urine sample. Whole blood samples were analyzed for erythrocyte count, haemoglobin concentration, mean corpuscular volume, platelet count, leukocyte count, and realtive numbers of neutrophils, band neutrophils, lymphcytes, atypical lymphocytes, eosinophils, monocytes and basophils. Haematocrit, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentrations were calculated from the erythrocyte data. Serum samples were anlyzed for alkaline phosphatase, alanine aminotransferase, and aspartat aminotransferase activities, and urea nitrogen, creatinine, total protein, and cholesterol concentrations
Statistics:
For statistical analyses, a one-way analysis of variance (ANOVA) and Bartlett's test were used at each sampling time. When the F-test from ANOVA was significant, the Dunnett test was used to compare means from the control group with each of the groups exposed to MA. test groups were compared with the appropriate control group by least significant difference and Dunnett's test when the ratio of vaiance (F) indicated significant amoug-to-within group variatins. Significance was judged at the .05 probability level.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
rats exposed to 750 ppm: hyperactivity, aggresion, hunched posture, lung noise, labored brathing, gasping, dry red nasal and ocular discharges, salivation, pallor, wet perineum, diarrhea, ruffled fur and facial hair loss.
Mortality:
mortality observed, treatment-related
Description (incidence):
rats exposed to 750 ppm: hyperactivity, aggresion, hunched posture, lung noise, labored brathing, gasping, dry red nasal and ocular discharges, salivation, pallor, wet perineum, diarrhea, ruffled fur and facial hair loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights for rats exposed to 750 ppm were sig. lower than the controls beginning on the third exposure day and persisting through the fourteenth day of recovery. No compound related changes were seen in rats exposed to 75 / 250 ppm.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative weights for a number of organs were signficantly lower in rats exposed to 75 ppm, but when these weights were adjusted for body weight, these differences became less although they remained statistically significant.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
After the tenth exposure, rats in the 750 ppm group hat increased erythrocyte, neutrophil, and monocyte counts, haemoglobin concentrations and haematocrit. increases in serum alanine and aspartate aminotransferase activities, as well as blood urea nitrogen
Details on results:
At the 750 ppm exposure levels, 4 rats died or were killed in extremis by the tenth exposure day. One rat also died after 12 d of recovery. All of these rats, together with 3 rats that were sacrificed following the tenth exposure, had similar gross and microscopic findings. These changes consisted of a distended gastrointestinal tract, small spleen and thymus due to depletion of lymphocytes, bone marrow hypocellularity, focal hepatic necrosis, dark of dull red lungs, and red nasal discharge. Nasal degeneration, severe necrosis leading to atrophy, and septal perforation were seen in the nasal turbinate mucosa.
In the 250 ppm exposure group, all rats examined after the tenth exposure had erosions and/or ulcerations of the nasal turbinate mucosa. No other lesions were seen in rats exposed to 250 ppm MA, and only mild nasal turbinate irritation was seen in rats expose to 75 ppm MA.
Dose descriptor:
LOAEC
Effect level:
75 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: local irritation in respiratory tract
Remarks on result:
other: corresponds to 96 mg/m3
Dose descriptor:
LOAEC
Effect level:
250 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: corresponds to 322 mg/m3
Critical effects observed:
not specified

Four rats in the 750 ppm group were found dead of were sacrificed in extermis on the eighth of tenth exposure day.

Although a red nasal discharge was seen in all groups, its severity was greater among rats exposed to 250 or 750 ppm.

No other signs of response to MA were seen in rats exposed to 250 ppm, and no unusual outward signs were seen in rats exposed to 75 ppm. A clear nasal discharge was also observed in rats from all treatment groups (including the controls) with equal severity and incidence.

During the 14 -d recovery period, lung noise, clear or dry red nasal discharges, ocular opacity, pallor, diarrhea, and discolored fur were observed in rats exposed to 750 ppm.

One additional rat from the 750 ppm group died on the twelfth day of the recovery period. No abnormal clinical signs were seen in any of the other groups of rats during the recovery period.

Conclusions:
It is concluded that MA can produce changes in the upper respiratory tract of the rat following repeated exposure to 250 ppm. Higher concentrations were not well tolerated and led to death, liver damage, and changes in the hematopoetic system. Exposure to 75 ppm approached the no-observed-adverse-effect level under these test conditions. Based on the results of this study, 75 ppm is the LOAEC for local effects in the respiratory tract, whereas 250 ppm is the LOAEC for systemic effects. The durrent TLV of 10 ppm for MA, based on analogy to ethylamine (ACGIH, 1986), appears to be appropriate and protective.
Executive summary:

In a study performed by Kinney et al. in 1990, methylamine produced only a mild irritation of the nasal turbinate mucosa in rats when exposed to 75 ppm for 10 days. Exposure to 250 ppm produced damage, restricted to the respiratory mucosa of the nasal turbinates of the rats (focal erosion and/or ulceration). Exposure to 750 ppm for 10 days led to severe body weight losses, clinical pathologic changes suggestive of liver damage, nasal degenerative changes, and haematopoietic changes, not all of which were reversible during the recovery period. Repeated exposure to 250 ppm MA produced focal erosion and/or ulceration of the nasal turbinate mucosa after the tenth exposure. This suggests that the material is absorbed in the anterior portion of the upper respiratory tract. No signs of focal or diffuse irritation were seen further into the respiratory tract, including the trachea and the lungs. All clinical chemical, haematologic, and anatomic pathologic findings, with the exception of the elevated blood urea nitrogen values, were normal after the recovery period. It thus appears that 250 ppm MA is relatively well tolerated by rats following 10 exposures and that the target tissue is the respiratory mucosa of the nasal turbinates. The lesion produced by this exposure regimen is mild but not reversible. Repeated exposure to 750 ppm was not well tolerated by rats. A number of mortalities occurred late in the exposure period, and one rat died late in the recovery period. An immediate response was reflected by weight loss, which persisted throughout the recovery period. Severe distention of the gastrointestinal tract, discolored lungs, nasal degeneration, and red nasal discharge were the predominant signs in rats either found dead, killed in extremis, or sacrificed after the tenth exposure. Lymphoid depletion in the thymus, spleen, and bone marrow is interpreted as being a secondary effect reflecting the stress of the exposure (severe weight loss) rather than a specific response to MA. The increased serum cholesterol concentration and alanine and aspartate aminotransferase activities and decreased serum protein concentration reflect the effect of MA on both the integrity (enzyme alterations) and function (protein, cholesterol) of the hepatic tissues. The increased red cell count, haematocrit, and haemoglobin concentration probably reflect a mild treatment-related absolute polycythaemia. This is consistent with relative polycythaemia (haemoconcentration), which may explain the increased BUN immediately following exposure and at the end of the recovery period. The increased numbers of neutrophils and monocytes and the increased acidity of the urine after the tenth exposure appear to be related to MA exposure. It is concluded that MA can produce changes in the upper respiratory tract of the rat following repeated exposure to 250 ppm. Higher concentrations were not well tolerated and led to death, liver damage, and changes in the haematopoetic system. Exposure to 75 ppm approached the no-observed-adverse-effect level under these test conditions. Based on the results of this study, 75 ppm is the LOAEC for local effects in the respiratory tract, whereas 250 ppm is the LOAEC for systemic effects. The current TLV of 10 ppm for MA, based on analogy to ethylamine (ACGIH, 1986), appears to be appropriate and protective.

For calculation of the derived no effect levels a LOAEL of 75 ppm (96 mg/m³) for local effects and a LOAEL of 250 ppm (322 mg/m³) for systemic effects, extracted out of the above mentionned clinical findings is used further for the risk characterization

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
96 mg/m³
Study duration:
subacute
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

Repeated dose toxicity: oral

In 1990, Sarkar et al. investigated the short- and long-term residual effects of methylamine, after oral gavage to rats. Methylamine did not cause adverse effects if administered either by gavage or in diet to rats during 90 days. The test material was administered to the rats by gavage (10 mg/kg bw) daily and in diet (100 mg/kg bw), which is a more relevant appliction route for humans. The rats received the test material during 21, 45, 65 and 90 days.

Body weight gains, organs weights and enzyme activities tested were similar to those of controls.The NOAEL identified was 10 mg/kg/day of methylamine.

In the OECD 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Rats, a daily gavage administration of 0, 250, 500, and 1000 mg/kg/day of the test substance Methylammonium chloride, to male and female rats for 98 to 119 consecutive days, resulted in increased liver and kidney weights, squamous metaplasia of the tracheal mucosa, and mucoid metaplasia of the glandular gastric mucosa. The increased liver weights in males (≥ 250 mg/kg/day) and females (≥ 500 mg/kg/day) and kidney weights in both sexes (1000 mg/kg/day) were consistent with pharmacological enzyme induction and were considered to be non-adverse. Minimal to mild squamous metaplasia of the tracheal mucosa and minimal focal mucoid metaplasia of the gastric glandular mucosa of several high-dose rats of both sexes were interpreted to be the result of topical exposure of the test substance Methylammonium chloride. The metaplastic changes in both tissues were interpreted to be adaptive and probably reversible. Therefore, both the tracheal and gastric metaplasia were considered to be non-adverse. There were no test substance-related effects on cause of death, gross pathology, or reproductive failure in adult rats. Gross examination of nursing pups that died before day 4 of lactation did not reveal any test substance-related findings. Under the conditions of this study, the no-observed-effect level (NOEL) for pathology for male and female rats was the highest dose tested, 1000 mg/kg/day.

Under the conditions of this study, the no-observed-effect level (NOEL) for systemic and reproductive toxicity was 500 mg/kg/day Methylammonium chloride based on reductions in parental body weights and food consumption and effects on reproductive outcome (reduced corpora lutea and subsequent reductions in implantations and litter size).

Repeated dose toxicity: dermal

No dermal long-term studies are available for methylamine. Such study is considered to be unnecessary because of animal welfare. (The test material is corrosive to the eyes, respiratory tract and stomach in acute studies).

Repeated dose toxicity: inhalation

Sriramachari S. and Jeevaratnam K. exposed rats to 19 µmol/L methylamine by inhalation and to 5.75 mmol/kg subcutaneous up to 10 weeks (Sriramachari and Jeevaratnam, 1994). No NOAEC could be identified because of only one dose level tested. A peculiar and extensive edema was observed at the end of 1 week in the rats exposed to MA vapours. The lungs were pale and had a washed out appearance. A moderate to severe interstitial pneumonitis without edema was noticed in lungs of rats exposed to MA by both routes after four weeks of exposure. In both the inhalation and subcutaneous groups, the lungs showed severe interstitial pneumonitis extending into peribronchial and perivascular areas at the end of 10 weeks of exposure.

In a study performed by Kinney et al. in 1990, methylamine produced only a mild irritation of the nasal turbinate mucosa in rats when exposed to 75ppm for 10 days. Exposure to 250 ppm produced damage, restricted to the respiratory mucosa of the nasal turbinates of the rats (focal erosion and/or ulceration). Exposure to 750 ppm for 10 days lead to severe body weight losses, clinical pathologic changes suggestive of liver damage, nasal degenerative changes, and haematopoietic changes, not all of which were reversible during the recovery period. Repeated exposure to 250 ppm MA produced focal erosion and/or ulceration of the nasal turbinate mucosa after the tenth exposure. This suggests that the material is absorbed in the anterior portion of the upper respiratory tract. No signs of focal or diffuse irritation were seen further into the respiratory tract, including the trachea and the lungs. All clinical chemical, haematologic, and anatomic pathologic findings, with the exception of the elevated blood urea nitrogen values, were normal after the recovery period. It thus appears that 250 ppm MA is relatively well tolerated by rats following 10 exposures and that the target tissue is the respiratory mucosa of the nasal turbinates. The lesion produced by this exposure regimen is mild but not reversible. Repeated exposure to 750 ppm was not well tolerated by rats. A number of mortalities occurred late in the exposure period, and one rat died late in the recovery period. An immediate response was reflected by weight loss, which persisted throughout the recovery period. Severe distention of the gastrointestinal tract, discolored lungs, nasal degeneration, and red nasal discharge were the predominant signs in rats either found dead, killed in extremis, or sacrificed after the tenth exposure. Lymphoid depletion in the thymus, spleen, and bone marrow is interpreted as being a secondary effect reflecting the stress of the exposure (severe weight loss) rather than a specific response to MA. The increased serum cholesterol concentration and alanine and aspartate aminotransferase activities and decreased serum protein concentration reflect the effect of MA on both the integrity (enzyme alterations) and function (protein, cholesterol) of the hepatic tissues. The increased red cell count, haematocrit, and haemoglobin concentration probably reflect a mild treatment-related absolute polycythaemia. This is consistent with relative polycythaemia (haemoconcentration), which may explain the increased BUN immediately following exposure and at the end of the recovery period. The increased numbers of neutrophils and monocytes and the increased acidity of the urine after the tenth exposure appear to be related to MA exposure. It is concluded that MA can produce changes in the upper respiratory tract of the rat following repeated exposure to 250 ppm. Higher concentrations were not well tolerated and led to death, liver damage, and changes in the haematopoetic system. Exposure to 75 ppm approached the no-observed-adverse-effect level under these test conditions. Based on the results of this study, 75 ppm is the LOAEC for local effects in the respiratory tract, whereas 250 ppm is the LOAEC for systemic effects. The current TLV of 10 ppm for MA, based on analogy to ethylamine (ACGIH, 1986), appears to be appropriate and protective.

For calculation of the derived no effect levels a LOAEL of 75 ppm (96 mg/m³) for local effects and a LOAEL of 250 ppm (322 mg/m³) for systemic effects, extracted out of the above mentioned clinical findings is used further for the risk characterization.


Repeated dose toxicity: via oral route - systemic effects (target organ) other: all gross lesions and masses

Justification for classification or non-classification

Classification is not warranted according to the criteria of EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No 1272/2008.