2-methyl-p-phenylenediamine

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 19, 2010 to March 9, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed by Japanese competent authorities according to GLP and equivalent OECD standardized guidelines.

Qualifier:

according to guideline

Guideline:

other: Testing Method for New Chemical Substances Alga growth inhibition test, Acute daphnia immobilization test and Acute fish toxicity test (21/11/2003; No.1121002, Pharmaceutical and Food Safety Bureau, Japanese Ministry of Health, Labour and Welfare)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Test waters were sampled from each test group. Samples for analysis were performed at T0, T24 (old solution), T72 (new solution) and T96 (old solution)- Sampling method and sample storage conditions before analysis: the samples were diluted arbitrarily with purified water (for those which are expected to be beyond the ragne of calibration curve); 0.75 mL sapmled and 0.75 mL acetonitrile added; mixing and dosing in LC/MS.

Vehicle:

no

Details on test solutions:

Storage method and confirmation of stability :During the test period, the test substance was stored in a refrigerating chamber for the use of storing test substances in this facility (storage conditions: cold storage, dark place, capped tightly).After the completion of the experiment, Infrared Absorption Spectrum of the stored test substance was measured. Since the obtained spectrum coincided with the one measured before the experiment, it was judged that the test substance was stable during the storage period. PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method: through dilution of a 1000 mg/L stock solution (direct addition of 20 mg/L in a 20 ML volumetric flask with dechlorinated tap water and mixed by turning upside down) with dechlorinated tap water. Test solutions were prepared in 5.0 L glass tanks, stirred by glass rod.- Eluate: no- Differential loading: no- Controls: dilution water- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not used- Evidence of undissolved material (e.g. precipitate, surface film, etc): No

Test organisms (species):

Oryzias latipes

Details on test organisms:

TEST ORGANISM- Common name: Himedaka- Source: Kitamura carp farm, 12-388 Gunchiku, Yatsushiro, Kumamoto Prefecture- Age at study initiation (mean and range, SD):Within a year from hatching- Length at study initiation (length definition, mean, range and SD): Recommended total length: 2.3 ± 1.2 cmMeasured value: mean value 2.44 cm (1.99-2.70 cm), n=10- Weight at study initiation (mean and range, SD):Measured value: mean value 0.107 g (0.057-0.145 g), n=10- Method of breeding:Breeding water: Dilution water (Tap water supplied by Yokohama city was treated under dechlorination process which includes the activated carbon filtration and the addition of Sodium thiosulfate (Dechlorinated tap water))Breeding type:Flow-through condition (1L/day or more per 1g of test fish)Water temperature: 24±1°CDissolved oxygen concentration: 80% or more of saturated oxygen concentrationpH: 6.0 – 8.5Irradiation condition: Room light, 16-hour light (1000 lux or less) / 8-hour darkType of feed: TetraMin, Tetra Feeding amount: ca.2% of fish body weight/day (No feeding for 24 hours before the beginning of the exposure)- Feeding during test: NoACCLIMATION- Acclimation period: January 25, 2010 – February 22, 2010- Acclimation conditions (same as test or not): yes except for food and flow-trough versus semi-static conditions in tests- Type and amount of food: see above- Feeding frequency:see above- Treatment with medicament during acclimation: Not done- Health during acclimation (any mortality observed): Mortality during the 7 days before the beginning of the exposure: Less than 5%QUARANTINE (wild caught) No- Duration:- Health/mortality:

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

45 mg/L CaCO3

Test temperature:

23.1-23.8°C

pH:

No adjustment; 7.0-7.6

Dissolved oxygen:

6.1 (minimum in old-24H-solution)-8.4

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 0.1, 0.18, 0.32, 0.56 and 1.0 mg/LMeasured initial concentrations: 0.0893, 0.177, 1.317, 0.519, 0.985 (arithmetic mean of "new solutions" T0, T72 new)Measured T24 (old): 0.00688 (8%), 0.0106 (6%), 0.0156 (5%), 0.0253 (5%), 0.0227 (2%)Measured T96 (old): 0.00709 (8%), 0.0146 (8%), other concentrations not measured because all the fishes were dead.Geometric mean (T0 and 24H): 0.025, 0.047, 0.070, 0.11, 0.15 mg/L

Details on test conditions:

TEST SYSTEM- Test vessel:glass tank (lid: teflon sheet)- Material, size, headspace, fill volume: volume of test vessel: 5L- Aeration: no aeration (but maintained at 60% or more of saturated oxygen concentration)- Type of flow-through (e.g. peristaltic or proportional diluter): / (semi-static)- Renewal rate of test solution (frequency/flow rate): 24H renewal- No. of organisms per vessel:10- No. of vessels per concentration (replicates):1- No. of vessels per control (replicates): 1- No. of vessels per vehicle control (replicates):/- Biomass loading rate: 1L or more per 1 g of fishTEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Tap water supplied by Yokohama city was treated under dechlorination process which includes the activated carbon filtration and the addition of Sodium thiosulfate (Dechlorinated tap water) to be used as dilution water. - Total organic carbon: 0.3 mg/L- Particulate matter: not detected- Metals: Hg, Cu, Cd, Zn, Pb, Ni, Cr, Mn, Sn, Ag, Co, Fe: not detected; Al: 0.02 mg/L- Pesticides: not detected- Chlorine: not detected- Alkalinity: 24 mg/L CaCO3- Ca/mg ratio: 13 mg/L Calcium; 3.1 mg/L magnesium- Conductivity:13 mS/m- Culture medium different from test medium: No- Intervals of water quality measurement: Mitsubishi Chemical Analytech Co., Ltd. is requested to measure the quality of the treated water periodically (at around every 6 months).OTHER TEST CONDITIONS- Adjustment of pH: No- Photoperiod: 16-hour light / 8-hour dark- Light intensity: Room light; 1000 lux or lessEFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Appearance of fishes was observed at 3, 24, 48, 72 and 96 hours after the beginning of the exposure. When dead fishes were found, they were removed immediately for preventing the deterioration of water. TEST CONCENTRATIONS- Spacing factor for test concentrations: 1.8- Justification for using less concentrations than requested by guideline:/- Range finding study: Yes- Test concentrations: 0.1, 1.0 and 10 mg/L; a second preliminary test was conducted, with the aim of confirming the toxicity of degraded product, with using test waters immediately after the preparation and at 24 hours after the preparation, at 0.1 and 1.0 mg/L.- Results used to determine the conditions for the definitive study: 0% mortality at 0.1 mg/L and 100% mortality at 1.0 mg/L in all the preliminary tests (and no abnormality observed in the living fishes).

Reference substance (positive control):

yes

Remarks:

Copper(II) sulfate pentahydrate, special grade chemical

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 0.032-0.27 mg/L

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.191 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 0.0893-0.317 mg/L

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.089 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

0.317 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Abnormality (abnormality in swimming) was observed in concentration group 2- Observations on body length and weight: /- Other biological observations:- Mortality of control: 0%- Other adverse effects control: No- Abnormal responses: /- Any observations : Appearances of control and concentration groups from 1 to 3 were colorless; however, those of concentration groups 4 and 5 were pale brown- Effect concentrations exceeding solubility of substance in test medium: floating/suspended solids and precipitation: not observed

Results with reference substance (positive control):

- Results with reference substance valid? Yes96-hour LC50 of the lot (date conducted acute toxicity study: January 21, 2009) = 0.56 mg/L (95% confidence limit 0.38 – 0.82 mg/L)96-hour LC50 of the past lots in this facility (since 1995, n=55):Mean ± S.D.: 0.82 ± 0.27 mg/LMinimum – maximum:: 0.33 – 1.5 mg/L

Reported statistics and error estimates:

LC50 calculated by binomial statistical method from concentrations 1 to 5.

Sublethal observations / clinical signs:

Discussion extracted from the report: Although the concentrations of the test substance in test waters at the time of preparation were demonstrated as being the same as the nominal values, they decreased to 1/10 or less in test waters immediately prior to renewal (or at the end of the exposure): hence, it was shown that the substance changed in its structure in water. Besides, the same degree of toxicities were observed in a preliminary test in which test fishes were exposed to test waters immediately after the preparation and at 24 hours after the preparation. Because of these facts, with regard to the toxicity observed in this study, although a possibility of deriving from the degraded product was considered, we could not reach to the conclusion that denies the possibility of deriving from the test substance. When calculating the results, if we use test substance concentrations which take account of concentration reduction such as time-weighted average concentration, it will be resulted in a result in which the toxicity effect of the degraded product is not reflected. Therefore in this study, with the aim of comprehensively assessing the toxicities of both test substance and degraded product, the arithmetic means of the measured concentrations at the time of preparing test waters, that is, the concentrations of the test substance before changing its structure, are used for calculating the results.

Other tests performed by the lead registrant on fresh and aged test solutions allowed to demonstrate that degradation/transformation processes continue to take place after 24H and lead to the formation of less toxic products. A re-calculation was therefore performed by the assessor based on the geometric mean measured concentrations at the beginning and the end of exposure (just after renewal and before renewal; arithmethic means) to consider the worst case situation and the higher toxicity of the parent compound.

Validity criteria fulfilled:

yes

Remarks:

The mortality in the control should not exceed 10 % at the end of the exposure (0%). The dissolved oxygen concentration must have been at least 60 % of the saturated oxygen concentration throughout the exposure period (6.1-8.4 g/L).

Conclusions:

96H-LC50= 0.05 mg/L based on geometric mean measured concentrations.

Executive summary:

The test substance 2,5 -diaminotoluene was tested on Oryzias latipes during 96H exposure in semi-static conditions (24H renewals). The test followed Japanese guidelines, equivalent to OECD guidelines, and GLP. Nominal concentrations were 0.1, 0.18, 0.32, 0.56 and 1.0 mg/L. Initial measured concentrations were almost the same as nominal values, but concentrations rapidly fell into solution to reach ranges of 2 to 8% of the nominal values at 24H after the renewal. 96H-LC50 reported in the study related to initial measured concentrations and was determined to be 0.191 mg/l. In agreement with the sponsor, and because tests performed on fresh and aged solutions revealed a higher toxicity of the parent compound, the 96H-LC50 value was re-calculated considering the geometric mean measured concentrations of the test substance at the beginning and at the end of the 24H period between each renewal. This was determined to be 0.05 mg/l. This study met all the validity criteria and was thus considered as reliable.

Description of key information

The test substance was tested on Oryzias latipes during 96H exposure in semi-static conditions (24H renewals). The test followed Japanese guidelines, equivalent to OECD guidelines, and GLP. Nominal concentrations were 0.1, 0.18, 0.32, 0.56 and 1.0 mg/L. Initial measured concentrations were almost the same as nominal values, but concentrations rapidly fell into solution to reach ranges of 2 to 8% of the nominal values at 24H after the renewal. 96H-LC50 reported in the study related to initial measured concentrations and was determined to be 0.191 mg/l. In agreement with the sponsor, and because tests performed on fresh and aged solutions revealed a higher toxicity of the parent compound, the 96H-LC50 value was re-calculated considering the geometric mean measured concentrations of the test substance at the beginning and at the end of the 24H period between each renewal. This was determined to be 0.05 mg/l. This study met all the validity criteria and was thus considered as reliable.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.05 mg/L