N-(carboxymethyl)-N-(phosphonomethyl)glycine

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From August 23, 1984 to February 21, 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to method equivalent or similar to OECD Guideline 413

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(carboxymethyl)-N-(phosphonomethyl)glycine

EC Number:

227-824-5

EC Name:

N-(carboxymethyl)-N-(phosphonomethyl)glycine

Cas Number:

5994-61-6

Molecular formula:

C5H10NO7P

IUPAC Name:

2-[(carboxymethyl)(phosphonomethyl)amino]acetic acid

Details on test material:

- Name of test material (as cited in study report): Glyphosate Intermediate
- Physical state: Fine white powder
- Lot/batch No.: LUIG 03014

Test animals

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
- Age at study initiation: 49 d
- Housing: Caged individually in suspended wire mesh cages
- Diet: Purinae Certified Rodent Chow # 5002, ad libitum
- Water: Tap water
- Acclimation period: 14 d


ENVIRONMENTAL CONDITIONS
- Photoperiod: 12 h light/12 h dark cycle


IN-LIFE DATES: From Aug. 23, 1984 to Nov. 15, 1984

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: 97% of the aerosol was less than 9 microns, in terms of aerodynamic diameter. Thus, an EAD of about 3 mcm with a GSD of 1.6 would describe an aerosol where essentially all (99%) of the aerosol was respirable (<10 mcm). See 'table 1' in 'Any other information on material and method' section.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 16 m3 glass and stainless steel exposure chambers
- Source and rate of air: Chamber ventilation air was supplied from an HVAC system
- Method of conditioning air: The air was filtered to remove particulates, and temperature and humidity were controlled.
- System of generating particulates/aerosols: Dust aerosol atmosphere of test material generated with an auger dust feed.
- Temperature, humidity, pressure in air chamber: Relative humidity was 51, 56, 49 and 59% in test group I, II, III and IV respectively; Chamber temperature was 71, 70, 69 and 71 °F in test group I, II, III and IV respectively.
- Air flow rate: 2000 and 4000 L/minute
- Method of particle size determination: The particle size distribution of the test material aerosol was determined once every other wk for each exposure group with an Andersen 8-stage cascade impactor



TEST ATMOSPHERE
- Brief description of monitoring of exposure concentration: Nominal exposure concentrations were determined weekly. Actual exposure concentrations were determined daily using pseudo average gravimetric techniques. In addition, real-time monitoring, using a light-scattering aerosol monitor, was performed hourly to assist in monitoring the operation of the generation system.
- Test material distribution in chamber atmosphere: Distribution of the test material within the chambers was entirely adequate for the purposes
of the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Once each wk, amount of test material collected on the glass fiber filters was determined analytically with high pressure liquid chromatography for the determination of exposure concentration. Distribution of the test material within the chambers was entirely adequate for the purposes of the study.

Duration of treatment / exposure:

13 wk

Frequency of treatment:

6h/d, 5d/wk for 13 wk

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 1.1, 10 or 105 mg/m3
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 0.86, 9.6 and 102 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

25 male and 20 female

Control animals:

yes

Details on study design:

- Dose selection rationale: Exposure levels were selected on the basis of pilot study. 5 males and 5 females albino rats (Charles River CD) were exposed for 6 h/d for 5 d to dust aerosol atmospheres of test material at a concentration of 0, 1.0, 10 or l00 mg/m3. No rats died and no clinical signs were seen in any animal. The high exposure level of 100 mg/m3 was considered acceptable for 13 wk inhalation study since it represented l0 times the ACGIH TLV for a nuisance dust (10 mg/m3 , total dust), and produced no significant toxicity in the pilot study.
- Rationale for animal assignment: Animals were randomly allotted to four groups by use of computer-generated random number tables

Positive control:

Not reported

Examinations

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: Twice daily (prior to, and again after exposure)

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once each wk


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

ORGAN WEIGHT: Yes
- Organs weighed: Adrenal, brain, heart, kidney, liver, lung and trachea, ovary and testis

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption of the males was determined only during the first 10 wk, prior to their cohabitation with the females. The food consumption of the females was measured for 13 wk.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Performed on all rats prior to study initiation and on ten animals/sex/ group after 6 and 13 wk of exposure
- Dose groups that were examined: All


HAEMATOLOGY: Yes
- Time schedule: Haematology evaluated once prior to study initiation on 5 animals/sex and also for 10 rats/sex/group at the 6 wk interim and 13 wk terminal sacrifice
- Parameters examined: Erythrocyte and total leukocyte counts, hematocrit, hemoglobin, platelet and reticulocyte counts, differential leukocyte count, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration


CLINICAL CHEMISTRY: Yes
- Time schedule: Clinical chemistry evaluated once prior to study initiation on 5 animals/sex and also for 10 rats/sex/group at the 6 wk interim and 13 wk terminal sacrifice
- Parameters examined: (sodium, potassium, chloride, calcium, phosphorous, alkaline phosphatase, total bilirubin, gamma glutamyltranspeptidase, aspartate aminotransferase, alanine aminotransferase, creatine phosphokinase, ornithine carbamyltransferase, urea nitrogen, creatinine, albumin, total protein, globulin, cholesterol, and glucose

Sacrifice and pathology:

SACRIFICE: Interim and terminal sacrifices were conducted after approximately 6 and 13 wk of exposure respectively. All survivors were euthanized by intraperitoneal sodium pentobarbital followed by exsanguination

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, tissues were processed for the preparation and microscopic examination of hematoxylin and eosin stained paraffin sections. A full tissue complement was prepared· for all animals in the control and high dosage groups and all animals which died during the course of the
study. In addition, sections. were prepared of the lungs, trachea, nasal tissue and all gross lesions for all animals.

Other examinations:

None

Statistics:

- Comparisons were made by the analysis of variance (one-way classification) and Bartlett's test for homogeneity of variance.
- If the group variances were homogeneous (p<0.05) then the appropriate t-test for equal variances as described by Steel and Torrie was performed using Dunnett's multiple comparison table to judge significance of differences.
- When appropriate, the data were analysed by non-parametric methods using the Conover and Iman rank transformation method

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: There were no exposure-related toxic signs. Five animals died during the study. One high dose (group IV) female died accidentally during wk eleven. Two females (one mid and one high exposure level) during study wk six and one male and one female from the high exposure group during study wk eleven were found dead. Excluding the accidental death, the other deaths were considered exposure-related since they occurred in the intermediate and high level exposure groups

BODY WEIGHT AND WEIGHT GAIN: Body weights of high exposure level males and females were consistently lower than the control group beginning at wk 2 or 3. At study termination high exposure level males and females weighed 13% and 11% less than controls, respectively.


FOOD CONSUMPTION: When compared to the control group on a g/animal/d basis, food consumption was decreased in Group IV males and females throughout the study. The differences were statistically significant at Week 1-6 and 9 for males and 1-6, and 9-11 for females. When considered on g/kg/d basis, food consumption was decreased in Group IV males and females for the first 4-5 wk of exposure; for the remainder of the study, food consumption was comparable to the control group. This depressed food consumption in the high dose level reflect some inappetence prior to acclimating to the exposure conditions.


OPHTHALMOSCOPIC EXAMINATION: No treatment related signs were observed.


HAEMATOLOGY: Exposure related elevation in erythrocyte count occured in group IV males (after 6 and 13 wk of exposure) and females (after 13 wk of exposure) However, This elevation observed cannot be explained from the data available. Hematocrit and hemoglobin concentrations of the Group IV females were also significantly increased at the 13 wk interval but this increase was not statistically significant at Week 6 when compared with control. In Group IV males hematocrit and hemoglobin levels appeared to be slightly increased similar to the females, but the differences were not statistically significant. The dose-related elevated white cell count, with a neutrophilic shift, seen particularly in females at the 6 wk interim sacrifice, were suggestive of a transient inflammatory process probably resulting form pulmonary deposition of the test material.

CLINICAL CHEMISTRY: Small increase in ALT levels in the females at 13 wk was observed but the differences were statistically significant for Groups III and IV when compared to control. Chloride levels were significantly decreased, compared to the controls, at 6 and 13 wk in the Group IV females and at 13 wk in the Group IV males. None of the serum biochemical differences observed were considered exposure-related, because the changes were quite small and there was no indication of a consistent time- or dose-related response.


ORGAN WEIGHTS: Test material related organ weight increases were observed in the lung/trachea. At the 6 wk interim sacrifice, these increases occured in male rats from Groups III and IV and in female rats from Groups Ill and IV. At the 13 wk terminal sacrifice, these increases occurred in male and female rats from Groups II, III and IV. This increased lung weight was probably related to deposition of test material and the resulting inflammatory
reaction. Apart from the above-mentioned changes none of the other organs showed any test material related changes in histopathology and hence the weight variations observed in other organs such as heart, brain and kidney were not considered toxicologically significant.


GROSS PATHOLOGY: There were no test material related macroscopic changes among any of the male or female rats that were sacrificed at the 6
wk interim, terminally sacrificed after 13 wk exposure to the test material or died during the course of study.


HISTOPATHOLOGY: NON-NEOPLASTIC: Test material related microscopic changes were observed in the respiratory system - trachea, bronchi, bronchioles, lung and nasal passages. At the 6 wk interim sacrifice, these changes consisted of chronic tracheitis in Group IV; chronic bronchitis and
luminal exudate in Groups III and IV; an increase in the incidence of interstitial inflammatory cell infiltration of the lung in Groups III and IV; intraalveolar vacuolated macrophages in Groups III. and IV; subacute inflammation (rhinitis), goblet cell proliferation (in Group III only) and squamous metaplasia in the respiratory mucosa (epithelium) of the nasal passages in Groups III and IV; and atrophy and regeneration in the olfactory mucosa in Groups III and IV. At the 13 wk terminal sacrifice, the changes were similar in the bronchi and lung, but additionally, there was chronic bronchiolitis in male rats from Group IV. The inflammatory reaction in the respiratory mucosa (of the nasal passage) persisted, but had a greater incidence of squamous metaplasia and hyperplasia, while the atrophic olfactory mucosa showed practically no regenerative activity. These effects were considered to be irritant responses due to direct contact of the test material with the respiratory passages. As such, they were not considered to represent systemic toxic effects resulting from absorption of the test material.

Effect levels

Dose descriptor:

NOEL

Effect level:

0.86 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Overall effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Thirteen week repeated exposure of the test material to Sprague-Dawley rats resulted in chronic inflammatory changes in the respiratory tract along with squamous metaplasia, hyperplasia and atrophic olfactory mucosa. As such, they were not considered to represent systemic toxic effects
resulting from absorption of the test material and probably resulted from the irritant effects on repeated inhalation exposure. Based on the test results, the low level of 0.86 mg/m3 was considered a no observed effect level (NOEL) for all parameters evaluated.

Executive summary:

A study was conducted to evaluate the repeated inhalation toxicity of test material in Sprague-Dawley rat.

 

25 males and 20 females were exposed to test material (as dust aerosol) at concentrations of 0, 1.1, 10 or 105 mg/m3 of air (mean analytical concentrations 0.86, 9.6, and 102 mg/m3 of air) for 6 h/d, 5 d/wk for 13 wk. The control group was exposed to room air only.

 

Interim and terminal sacrifices were conducted after approximately 6 and 13 weeks of exposure, respectively. No significant exposure-related toxic signs were observed. Depressed body weight gain in both males and females exposed to 105 mg/m3 was evident after approximately two weeks of exposure and became progressively more pronounced as the exposures continued. A transient depression in food consumption was observed during the first five or six weeks of exposure in animals exposed to 105 mg/m3.

An elevated white cell count after six weeks of exposure to 10 and 105 mg/m3 probably indicated a transient inflammatory response to pulmonary deposition of test material. An elevated red cell count was observed after 13 weeks of exposure to 105 mg/m3. All other hematologic parameters and all serum biochemical parameters evaluated were similar to the control values.

No exposure-related abnormalities were found during the ophthalmologic examinations.

Macroscopic observations at necropsy did not indicate any abnormalities related to the exposure conditions. Lung and trachea weights were elevated in, both males and females. The increase was seen at both 6 and 13 weeks and was clearly dose-related, with effects apparent in the low level at 13 weeks. This increased lung weight was probably related to deposition of test material and the resulting inflammatory reaction.

Microscopically, exposure-related effects were seen only in the respiratory system, and then almost exclusively in the intermediate and high level animals. Tracheitis, bronchitis and inflammation at the alveolar level was observed after 6 and13 weeks of exposure. These effects were considered to be irritant responses due to direct contact of the test material with the respiratory passages.

Under the conditions of the test, the low level of 0.86 mg/m3 was considered to be a no observed effect level (NOEL) for all parameters evaluated.